Compositions for treating cancer with KRAS mutations and uses thereof

Abstract

The present application provides guide RNAs and genome-editing complexes or nanoparticles that are useful for specifically targeting a mutated KRAS. Exemplary genome-editing complexes or nanoparticles comprise cell-penetrating peptides, and optionally a DNA nuclease (such as Cas9) or a polynucleotide encoding the DNA nuclease.

Description

COMPOSITIONS FOR TREATING CANCER WITH KRAS MUTATIONS AND USESTHEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to international application No. PCT / US2024 / 019288, filed March 9, 2024, the contents of which are incorporated by reference in their entirety for all purposes.FIELD OF THE APPLICATION

[0002] The present application relates to guide RNAs and genome-editing complexes or nanoparticles that are useful for specifically targeting a mutated KRAS.BACKGROUND OF THE APPLICATION

[0003] The RAS subfamily member KRAS is the most frequently mutated oncogene in cancers, including highly lethal lung, colon, and pancreatic cancers (Cox et al. 2014 Nat Rev Drug Discov 13, 828). Activating mutations in KRAS play potent roles in cancer initiation, propagation, and maintenance, representing important therapeutic targets (Cox et al. 2014). A common cancer-associated mutation occurs in KRAS at the glycine-encoding codon- 12. Specifically, the single-nucleotide missense substitutions c.35 G > T and c.35 G > A replace glycine at position 12 with valine (G12V) and aspartic acid (G12D), respectively. G12V and G12D substitutions are among the most commonly observed mutations in pancreatic adenocarcinoma (30% and 51%, respectively) and colorectal adenocarcinomas (27% and 45%, respectively) and have been associated with poor prognosis (Jones, S. et al. 2008, Science 321, 1801; Wood, L. D. et al. 2007, Science 318, 1108).

[0004] Today there is an urgent need of a potent strategy to target KRAS mutations. Although several attempts have been made to target RAS-dependent cancers, the direct inhibition of RAS proteins has not been successful and most of the approaches affected the downstream effectors of mutated RAS. The recent development of KRAS (G12C)- specific inhibitors (Patricelli et al. 2016 Cancer Discov 6: 316) and of a non-mutant selective RAS- binding domain inhibitor (Athuluri-Divakar et al.2016. Cell 165: 643), showed the potential of direct targeting mutated RAS oncogenes. Directly targeting mutated RAS oncogenes has the potential to disrupt the functions of both the aberrant RAS proteins and their downstream effector pathways. However, producing this chemical is challenging due to its complex structure and mutation- specific inhibition was not achieved using small molecules for KRAS (G12D) or KRAS (G12V), which occur more frequently than KRAS (G12C). KRAS silencingusing small interfering RNAs (siRNAs) that selectively inhibit mutant KRAS mRNAs have also been reported, but considering the continuous expression of KRAS mutant, permanent delivery is required for target RNA suppression (Zorde Khvalevsky et al. 2013 Proc Natl Acad Sci 110: 20723) to maintain a complete knockdown (Brummelkamp et al. 2002 Cancer Cell 2: 243).

[0005] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.BRIEF SUMMARY OF THE APPLICATION

[0006] The present application in one aspect provides non-naturally occurring polynucleotide comprising a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, 273- 341. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 273-341. In some embodiments, the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341. In some embodiments, the guide RNA specifically targets G12C, and the target sequence is set forth in SEQ ID NO: 273. In some embodiments, the guide RNA specifically targets G12R, and the target sequence is set forth in any one of SEQ ID NOs: 274-283. In some embodiments, wherein the target sequence is set forth in SEQ ID NO: 274, 277, or 279. In some embodiments, the guide RNA specifically targets G12A, and the target sequence is set forth in any one of SEQ ID NOs: 284-294. In some embodiments, the target sequence is set forth in SEQ ID NO: 284, 285, or 290. In some embodiments, the guide RNA specifically targets G12S, and the target sequence is set forth in any one of SEQ ID NOs: 295-304. In some embodiments, the target sequence is set forth in SEQ ID NO: 295, 298, or 300. In some embodiments, the guide RNA specifically targets G13D, wherein the target sequence is set forth in any one of SEQ ID NOs: 305-309. In some embodiments, the target sequence is set forth in SEQ ID NO: 305 or 308. In some embodiments, the guide RNA specifically targets G13C, wherein the target sequence is set forth in any one of SEQ ID NOs: 310-315. In some embodiments, the target sequence is set forth in SEQ ID NO: 310 or 313. In some embodiments, the guide RNA specifically targets Q61H, wherein the target sequence is set forth in any one of SEQ ID NOs: 316-322. In some embodiments, the target sequence is set forth in SEQ ID NO: 316 or 321. In some embodiments, the guide RNA specifically targets Q61L, wherein the target sequence is set forth in any one of SEQ ID NOs: 323-329. In someembodiments, the target sequence is set forth in SEQ ID NO: 323 or 328. In some embodiments, the guide RNA specifically targets A18D, wherein the target sequence is set forth in any one of SEQ ID NOs: 330-332. In some embodiments, the target sequence is set forth in SEQ ID NO: 332. In some embodiments, the guide RNA specifically targets K117N, wherein the target sequence is set forth in any one of SEQ ID NOs: 333-335. In some embodiments, the target sequence is set forth in SEQ ID NO: 333. In some embodiments, the guide RNA specifically targets A146T, wherein the target sequence is set forth in any one of SEQ ID NOs: 336-341. In some embodiments, the target sequence is set forth in SEQ ID NO: 336 or 339.

[0007] In some embodiments according to any of the non-naturally occurring polynucleotides described above, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).

[0008] In some embodiments according to any of the non-naturally occurring polynucleotides described above, the polynucleotide a) is chemically modified (e.g., 5-Methoxyuridine ), and / or b) comprises prime editing guide RNA (pegRNA). In some embodiments, the pegRNA comprises a prime editing site and a reverse transcriptase template.

[0009] The present application in another aspect provides genome-editing complexes comprising a) any of the polynucleotides described above, and b) a carrier, wherein the carrier promotes the delivery of the polynucleotide to a cell. In some embodiments, the complex further comprises a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR- associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof. In some embodiments, the DNA nuclease comprises a Cas polypeptide. In some embodiments, the Cas polypeptide a) is or comprises a Cas9 or a modified Cas9 nuclease, and / or b) is fused with a deaminase. In some embodiments, the deaminase is a cytosine base editor or an adenine base editor. In some embodiments, the cytosine base editor is BE3, BE4, or A3A-PBE. In some embodiments, the adenine base editor is ABE7.10 or ABE8e.

[0010] In some embodiments according to any of the genome-editing complex described above, the carrier comprises a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a cell-penetrating peptide (CPP). In some embodiments, the carrier comprises aCPP. In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the cellpenetrating peptide further comprises one or more moieties covalently linked to N-terminus of the first cell-penetrating peptide, and wherein the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety, optionally wherein: a) the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide; b) the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide, further optionally wherein the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351-352, 356-366, 378-386, 397-407, and 423-426. In some embodiments, the targeting moiety is conjugated to beta-alanine of an VEPEP-6 peptide (e.g., SEQ ID NO: 89 or 90). In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx, optionally wherein the linker moiety is a PEG moiety or Ava, further optionally wherein the PEG moiety comprises any of two to twelve, two to ten, two to seven and two to three ethylene glycol units. In some embodiments, the cell-penetrating peptide comprises, from N- terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N- terminus of the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide a) further comprises a carbohydrate moiety, optionally wherein the carbohydrate moiety is GalNAc, b) is a retro-inverso peptide, and / or c) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, 153-175, 259-270, 272, 353- 355, 367-377, 382-383, 387-396, 418-422, and 427-434, optionally wherein the CPP comprises an amino acid selected from the group consisting of 89, 90, 162, 270, 355, 427- 434. In some embodiments, the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., about 1: 1 to about 60:1, about 1:1 to about 1:50:1, about 1:1 to about 40:1, about 1:1 to about 30:1, about 1:1 to about 20:1), optionally wherein the molar ratio of the cell -penetrating peptide to the guide RNA is about 20:1. In some embodiments, the complex comprises a polynucleotide encoding a Cas nuclease, and wherein the molar ratio of the cell -penetrating peptide to the polynucleotide encoding the DNA nuclease is between about 1:1 and about 80:1, further wherein the Cas nuclease is a Cas9 or amodified Cas9, further wherein the molar ratio of the cell-penetrating peptide to the polynucleotide encoding the DNA nuclease is 20:1.

[0011] In some embodiments according to any of the genome-editing complex described above, the genome-editing complex further comprises one or more additional guide RNAs comprising different guide sequences, optionally wherein at least two of the two or more guide RNAs target one single KRAS mutation, further optionally wherein at least two of the two or more guide RNAs target two or more different KRAS mutations, further optionally wherein at least two of the two or more guide RNAs target G12D, G12V, G12C, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, KI 17N, or A146T.

[0012] In some embodiments according to any of the genome-editing complex described above, the average diameter of the genome-editing complex is between about 10 nm and about 300 nm.

[0013] In some embodiments according to any of the genome-editing complex described above, the complex comprises a) a first CPP comprising an amino acid sequence set forth in any of SEQ ID NO: 89, 90, 270, 153-155, 434 and 435, optionally wherein the first CPP comprises an amino acid sequence set forth in SEQ ID NO: 434 or 435, and b) a second CPP comprising an amino acid sequence set forth in any of 427-433, optionally wherein the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429.

[0014] The present application in another aspect provides nanoparticles comprising a core comprising the genome-editing complex described above.

[0015] The present application in another aspect provides pharmaceutical compositions comprising any of the guide RNAs described above, any of the genome-editing complex described above, or any of the nanoparticles described above, and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.

[0016] The present application in another aspect provides methods of preparing any of the genome-editing complex described above, comprising combining the first cell-penetrating peptide with the guide RNA, thereby forming the genome-editing complex.

[0017] The present application in another aspect provides methods of modifying mutated KRAS in a cell, comprising contacting the cell with any of the guide RNAs, any of the genome-editing complexes, or any of the nanoparticles. In some embodiments, the methodresults in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 20%- about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in the cell.

[0018] The present application in another aspect provides methods of delivering a guide RNA to a cell, comprising contacting the cell with any of the guide RNAs, any of the genome-editing complexes, or any of the nanoparticles. In some embodiments, the method results in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 20%- about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in the cell.

[0019] The present application in another aspect provides methods of treating a cancer in an individual comprising administering the individual an effective amount of any of the pharmaceutical compositions. In some embodiments, the individual comprises a secondary mutation in KRAS, optionally wherein the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation. In some embodiments, the cancer comprises a copy number variation in KRAS. In some embodiments, the cancer comprises an upregulated KRAS mRNA level and / or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy. In some embodiments, the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter. In some embodiments, the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and / or N-RAS. In some embodiments, the individual has been subjected to a KRAS inhibitor treatment. In some embodiments, the cancer is resistant, refractory or recurrent to the KRAS inhibitor, further optionally the individual developed a secondary mutation after the KRAS inhibitor treatment. In some embodiments, the KRAS inhibitor specifically binds to the mutant KRAS protein. In some embodiments, the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC-9805, sotorasib, adagrasib, ganetespib, RMC-6236, YL- 17231, BDTX-4933, QTX3034, ABT-200, ADT-1004, AN9025, OC211, JAB-23425, BI-2865, BI-2493, ABREV01, A2A-03, LY3537982, and LY-4066434. In some embodiments, the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC-9805, sotorasib,adagrasib, and ganetespib. In some embodiments, the individual does not develop a secondary KRAS mutation in any of the exons after the KRAS treatment. In some embodiments, the method of claim 30, further comprising administering a second agent. In some embodiments, the method results in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 20%-about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in cancer cells harboring a KRAS mutation (i.e., the KRAS mutation the guide RNA targets).BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGs. 1A-1B show proliferation rates of H358 cells and five stable sotorasib resistant H358 clones under the treatment of sotorasib or adagrasib at different concentrations. The five stable sotorasib resistant H358 clones are H-358-C1, H-358-C2, H358-C3 (KRAS G12C / Y96D), H-358-C4 (KRAS G12C / R68M) and H-358-C5 (KRAS G12C / A59T).Secondary mutations were identified in H358-C3 (KRAS G12C / Y96D), H-358-C4 ((KRAS G12C / R68M). and H-358-C5 (KRAS G12C / A59T). No secondary mutation was identified in the exon 2 to 3 of the KRAS gene of H358 -Cl and H358-C2.

[0021] FIG. 2A shows proliferation rates of H358 cells and five stable sotorasib resistant H358 clones under the treatment of an exemplary complex ADGN-122 that has a gRNA specifically targets G12C complexed with a cell-penetrating peptide. FIG. 2B shows a western blot result of the level of p-ERK in the H-358 and H-358 resistant clones cells after treatment of ADGN-122, sororasib or adagrasib. FIG. 2C shows relative levels of KRASG12Cgene expression in parental and AMG 510-resistant H-358 clones. FIG. 2D shows the ratio of KRASG12C-GTP in parental and AMG 510-resistant H-358 clones. The ratio is calculated as KRASG12Cover RAS over GAPDH.

[0022] FIG. 3 shows proliferation rates of H358 cells and three resistant H358 clones under the treatment of an exemplary complex that has siRNA specifically targeting G12C complexed with a cell-penetrating peptide.

[0023] FIG. 4 depicts a summary of IC50 of different treatments against H358 cells and H358 resistant clones.

[0024] FIG. 5A shows relative levels of KRAS gene expression in parental and MTRX-1133- resistant PANC-1 and ASPC-1 cells. FIG. 5B shows protein expression levels of KRASG12D, relative to GAPDH protein expression levels, in parental and MTRX-1133-resistant PANC-1and ASPC-1 cells. FIG. 5C shows the ratio of KRASG12D-GTP in parental and MTRX-1133- resistant PANC-1 and ASPC-1 cells as determined by a pulldown assay. The ratio is calculated as KRASG12Dover RAS over GAPDH.

[0025] FIG. 6A shows proliferation rates of parental and MTRX-1133-resistant PANC-1 and ASPC-1 cells under the treatment of an ADGN-123 nanoparticle that has a gRNA specifically targeting KRAS G12D associated with an ADGN peptide. FIG. 6B shows proliferation rates of parental and MTRX-1133-resistant PANC-1 and ASPC-1 cells under the treatment of MTRX-1133. FIG. 6C is a table of the IC50 of cell lines treated with either ADGN-121 or MTRX-1133. Lines labeled as PANC-1 and ASPC-1 are the parental cell lines. Cell lines denoted with Cl, C2, or C3 are the MTRX-1133-resistant cell lines.

[0026] FIG. 7 shows the expression efficiency of ADGN / Cas9mRNA / sgRNA complexes in PANC-1 cells. The complexes contained only ADGN-100, only ADGN-106, ADGN-100 mixed with the indicated targeting peptide (ADGN-100-hydro-3, ADGN-106-hydro-3, ADGN-1088, or ADGN-108-R91), or ADGN-106 mixed with the indicated targeting peptide (ADGN-100-hydro-3, ADGN-106-hydro-3, ADGN-1088, or ADGN-108-R91). The complexes were mixed with either 0.1 pg or 0.5 pg of CAS9 mRNA. CAS9 protein expression was measured by ELISA 24 hr post-transfection and compared to an LNP formulation.

[0027] FIG. 8 shows target sequences for the design of sgRNA targeting KRAS with a G12D, G12V, G12C, G12R, G12S, G12A, G13D, G13C, Q61H, Q61L, A18D, KI 17N, or A146T mutation.

[0028] FIG. 9 shows editing efficiency of various guide RNAs discussed in Example 9.DETAILED DESCRIPTION OF THE APPLICATION

[0029] The present application in one aspect provides novel guide RNAs that target specific KRAS mutant sequences (such as KRAS with a G12C, G12D, G12V, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T mutation). As demonstrated in the Examples, the exemplary guide RNAs were able to specifically target KRAS gene that bears a specific mutation while not affecting KRAS wildtype sequence.

[0030] The present application in another aspect provides genome-editing complexes comprising a) a cell-penetrating peptide, and b) a guide RNA described herein. As demonstrated in the examples, administration of exemplary genome-editing complexesincluding a cell-penetrating peptide and a guide RNA as described herein successfully treated individuals having tumors with KRAS mutations without inducing any significant toxicity, emergence of off target effects or other KRAS mutations. In some cases, one or two administrations of the exemplary genome-editing complexes resulted in a complete regression of tumors.

[0031] Also provided herein are nanoparticles comprising the genome-editing complexes, methods of preparing and using the guide RNAs, genome-editing complexes or nanoparticles as well as kits and articles of manufacture useful for the methods.I. Definitions

[0032] The term “guide RNA” refers to a polynucleotide that cleaves, inserts, or links a target DNA in a cell via RNA editing. The guide RNA may be a single-chain guide RNA (sgRNA). The guide RNA may be a CRISPR RNA (crRNA) specific to the target nucleotide sequence. The guide RNA may further include a trans-activating crRNA (tracrRNA) interacting with Cas9 nuclease. The tracrRNA may include a polynucleotide forming a loop structure. The guide RNA may have a length of 10 nucleotides to 30 nucleotides. The guide RNA may have a length of, for example, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

[0033] The guide RNA may include RNA, DNA, PNA, or a combination thereof. The guide RNA may be chemically modified (e.g., 5-Methoxyuridine ).

[0034] The guide RNA may be a component of molecular scissors (programmable nuclease). The molecular scissor refers to all types of nucleases capable of recognizing and cleaving a specific site on the genome. The molecular scissors may be, for example, transcription activator-like effector nuclease (TALEN), zinc-finger nuclease, meganuclease, RNA-guided engineered nuclease (RGEN), Cpfl, and Ago homolog (DNA-guided endonuclease). The RGEN refers to a nuclease including a guide RNA specific to a target DNA and Gas protein as components. The polynucleotide may be, for example, a component of RGEN.

[0035] In aspects of the application the term “single guide RNA” or “sgRNA” refers to a polynucleotide sequence comprising a guide sequence, a tracr sequence and a tracr mate sequence. The term “guide sequence” refers to the about 20 bp sequence within the guideRNA that specifies the target site. The term “tracr mate sequence” may also be used interchangeably with the term “direct repeat(s)”.

[0036] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0037] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.

[0038] The terms “non-naturally occurring,” “synthetic,” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0039] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, RNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including for example locked nucleic acid (LNA), unlocked nucleic acid (UNA), and zip nucleic acid (ZNA), which can be synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues ofnatural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and / or deoxyinosine residues (Batzer e al., Nucleic Acid Res., 19:5081 ( 1991); Ohtsuka et a ., j . Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylases, and alkylhalides. "Oligonucleotide," as used herein, generally refers to short, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0040] In general, “CRISPR system” refers collectively to proteins, transcripts and other molecules involved in the activity of CRISPR-associated (“Cas”) nucleases (such as RNA- guided endonucleases, or “RGENs”), including Cas gene products, Cas gene sequences, tracr (trans-activating CRISPR) sequences (e.g. tracrRNA or an active partial tracrRNA), tracr- mate sequences (including a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences, transcripts, and products derived from a CRISPR locus. In some embodiments, one or more molecules of a CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more molecules of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by molecules that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed tohave complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is present in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template,” “editing polynucleotide,” “editing sequence,” “donor sequence,” or “donor nucleic acid”. In aspects of the application, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the application the recombination is homologous recombination.

[0041] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more molecules of a CRISPR system are introduced into a host cell such that formation of a CRISPR complex at one or more target sites can occur. For example, a Cas nuclease, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be introduced into a host cell to allow formation of a CRISPR complex at a target sequence in the host cell complementary to the guide sequence.

[0042] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or othernon-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80% / , 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0043] As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993). Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”. Elsevier, N.Y.

[0044] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.

[0045] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referredto as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0046] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0047] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0048] As used herein, “treatment” or “treating” refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.

[0049] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0050] As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

[0051] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0052] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 10% to 70% should be considered to have specifically disclosed subranges such as from 10% to 30%, from 10% to 40%, from 10% to 50%, from 20% to 40%, from 20% to 60%, from 30% to 60% etc., as well as individual numbers within that range,. This applies regardless of the breadth of the range.

[0053] The compositions and methods of the present application may comprise, consist of, or consist essentially of the essential elements and limitations of the application described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful.

[0054] Unless otherwise noted, technical terms are used according to conventional usage.Guide RNAs

[0055] The complex or nanoparticle described herein comprises a guide RNA that targets a mutated KRAS, such as any of the guide RNA described in the “synthetic guide RNAs” section. In some embodiments, the mutated KRAS comprises one or more mutations selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, GBR, G13A, G13D, G13V, GBP, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T and A146V. In some embodiments, the mutated KRAS comprises one or more mutations selected from the group consisting of G12D, G12C, G12V, G12A, G12S, GBR, G13D and G13C.

[0056] In some embodiments, the guide RNA for targeting mutated KRAS described herein comprises a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, 271, and 273-341.

[0057] The present application in another aspect provides novel guide RNAs discussed below. In some embodiments, there is provided a polynucleotide (e.g., a non-naturally occurring polynucleotide) comprising a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequencesubstantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 272-341. In some embodiments, the guide RNA is a singleguide RNA (sgRNA). In some embodiments, the guide RNA is 100% complementary to the target sequence and has the same length as the target sequence.

[0058] The present application further provides a composition, a complex (e.g., any of the complexes described herein), a nanoparticle, or a pharmaceutical formulation comprising any of the guide RNAs described above. In some embodiments, the composition, complex, nanoparticle, or pharmaceutical formulation described herein comprising any one or more (e.g., two, three, four or more) of these guide RNAs.Guide RNA targeting G12C

[0059] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12C mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in SEQ ID NO: 273.

[0060] In some embodiments, the guide RNA comprises a nucleotide sequence 100% complementary to a target sequence of SEQ ID NO: 273.

[0061] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0062] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the guide sequence has a length of about24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0063] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 253. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0064] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 254. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0065] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 255. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0066] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0067] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0068] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0069] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 256. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0070] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0071] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 273. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0072] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 271. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0073] KRAS G12C mutation was present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting G12R

[0074] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12R mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 274-283.

[0075] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 274. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0076] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 275. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0077] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 276. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0078] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 277. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0079] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 278. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0080] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 279. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0081] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 280. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0082] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 281. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0083] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 282. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0084] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12R, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 283. In some embodiments, the guide sequence has a length ofabout 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0085] KRAS G12R is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting G12A

[0086] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12A mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 284-294.

[0087] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 284. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0088] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 285. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0089] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 286. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0090] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 287. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0091] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 288. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0092] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 289. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0093] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 290. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0094] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 291. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0095] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 292. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0096] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 293. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0097] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12A, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 294. In some embodiments, the guide sequence has a length ofabout 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0098] KRAS G12A is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting G12S

[0099] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G12S mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 295-304.

[0100] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 295. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0101] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 296. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0102] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 297. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0103] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 298. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0104] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 299. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0105] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 300. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0106] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ endor 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 301. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0107] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCC at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 302. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0108] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 303. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0109] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G12S, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of ACG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 304. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0110] KRAS G12S is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a 1malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting G13D

[0111] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G13D mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 305-309.

[0112] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 305. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0113] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 306. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0114] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 307. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0115] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequencecomplementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 308. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0116] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 309. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0117] KRAS G13D is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting G13C

[0118] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a G13C mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 310-315.

[0119] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 310. In some embodiments, the guide sequence has a length ofabout 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0120] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 311. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0121] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 312. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0122] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 313. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0123] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 314. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0124] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising G13C, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GCG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 315. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0125] KRAS G13C is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting Q61H

[0126] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a Q61H mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 316-322.

[0127] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 316. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0128] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 317. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0129] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 318. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0130] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 319. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0131] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 320. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0132] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 321. In some embodiments, the guide sequence has a length ofabout 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0133] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61H, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 322. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0134] KRAS Q61H is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting Q61L

[0135] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a Q61L mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 323-329.

[0136] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 323. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0137] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 324. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0138] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 325. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0139] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 326. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0140] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GA GT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 327. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0141] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 328. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0142] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising Q61L, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 329. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0143] KRAS Q61L is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting A18D

[0144] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a A18D mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 330-332.

[0145] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 330. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0146] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 331. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0147] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A18D, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TTG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 332. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0148] KRAS A18D is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting K117N

[0149] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a KI 17N mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 333-335.

[0150] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTGA at the 5 ’ endor 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 333. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0151] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 334. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0152] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 333. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0153] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising KI 17N, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CCT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 335. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0154] KRAS KI 17N is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is amalignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).Guide RNA targeting A146T

[0155] In some embodiments, there is provided a guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising a A146T mutation, wherein the guide sequence comprises a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 336-341.

[0156] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 336. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0157] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 337. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0158] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence, flanked by a PAM sequence of AGG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 338. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0159] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequencecomplementary to the target sequence flanked by a PAM sequence of AAG at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 339. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0160] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of TCA at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 340. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0161] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of GTT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 341. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0162] In some embodiments, the guide RNA (such as a single-guide RNA) for targeting a mutated KRAS comprising A146T, wherein the guide RNA comprises a guide sequence complementary to the target sequence flanked by a PAM sequence of CCT at the 5 ’ end or 3 ’ end of the guide RNA, and wherein the target sequence comprises the nucleotide sequence of SEQ ID NO: 335. In some embodiments, the guide sequence has a length of about 24-28 base pairs, 20-24 base pairs, 20-22 base pairs, 20-21 base pairs, 18-20 base pairs, or 15-18 base pairs.

[0163] KRAS A146T is present in various diseases (such as a solid cancer or a liquid cancer, such as myelodysplastic syndrome). Exemplary cancers include lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cancer, pancreatic ductaladenocarcinoma, multiple myeloma, and glioma. In some embodiments, the cancer is a malignant or advanced cancer. Guide RNA described herein can be used for treating any of the above diseases (such as via methods described herein).

[0164] In some embodiments, the guide RNA is in the form of RNA.

[0165] In other embodiments, the guide RNA is in the form of DNA encoding the RNA (i.e., gDNA). In some embodiments, the DNA is a plasmid DNA. In some embodiments, the plasmid DNA further comprises a DNA encoding a DNA nuclease (such as Cas9).

[0166] In some embodiments, the guide RNA further comprises a DNA nuclease recruiting sequence.

[0167] In some embodiments, the guide RNA is a single guide RNA (sgRNA) further comprising an auxiliary trans-activating crRNA (tracrRNA).

[0168] In some embodiments, the guide RNA further comprises a tracr mate sequence, a tracr sequence, and / or a tail sequence. In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the guide sequence, tracr sequence and tracr mate sequence are contained within a single RNA (referred to herein as a “single-guide RNA,” or “sgRNA”), such that hybridization between the tracr sequence and the tracr mate sequence produces a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternativesequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In some embodiments, the sgRNA has at least two or more hairpins. In some embodiments, the sgRNA has two, three, four or five hairpins. In some embodiments, the sgRNA has at most five hairpins. In some embodiments, the sgRNA further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides.

[0169] In some embodiments, the guide RNA is a prime editing guide RNA (pegRNA) that further comprises a primer binding sequence and / or a desired RNA sequence (for example at the 3 ’end of the guide RNA). PegRNA can form a complex with a prime editor (such as a fusion protein comprising a modified Cas9 protein and a reverse transcriptase), thereby allowing prime editing of targeted sequences. See for example, Anzalone & Liu et al., Nature. 2019 Dec;576 (7785): 149- 157.

[0170] In some embodiments, the guide RNA comprises one or more modification (e.g., chemical modification). . In some embodiments, the gRNA has one or more modified nucleotides, including nucleobase modification and / or backbone modification. Exemplary modifications to the guide RNA include, but are not limited to, phosphorothioate backbone modification, 2’ -substitutions in the ribose (such as 2’-O-methyl and 2’-fluoro substitutions), LNA, and L-RNA. In some embodiments, the guide RNA does not have modifications to the nucleobase or backbone.

[0171] In some embodiments, the guide RNA comprises a moiety that promotes the annealing of guide sequence. In some embodiments, the moiety comprises a synthetic nucleotide sequence, wherein the synthetic sequence is about 1-200 nucleotides, such as about 5 to about 100 nucleotides, such as about 8 to about 80 nucleotides, such as about 10 to about 50 nucleotides, such as about 12 to about 40 nucleotides.

[0172] In some embodiments, the guide RNA (such as a single-guide RNA) has a length of no more than about 200 nucleotides, such as about 5 to about 100 nucleotides, such as about8 to about 80 nucleotides, such as about 10 to about 50 nucleotides, such as about 12 to about 40 nucleotides.

[0173] Many delivery systems can be employed to deliver any of the genome-editing complexes guide RNAs, complexes, nanoparticles, and compositions described in this application, including but not limited to, viral, liposome, electroporation, microinjection andconjugation, to achieve the introduction of the gRNA into a host cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding gRNA of the present invention to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a construct described herein), naked nucleic acid, and nucleic acid complexed with a delivery carrier, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes for delivery to the host cell.

[0174] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or genegun, naked DNA and artificial virions.

[0175] The use of RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.Complexes

[0176] In some embodiments, there is provided genome-editing complexes comprising a) carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a cellpenetrating peptide (CPP)), and b) a polynucleotide comprising a guide RNA targeting mutated KRAS comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NO: 1-37, 241-257, 271, and 273-341 (e.g., . In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof. In some embodiments, the complex comprises both the guide RNA and an mRNA encoding a Cas9.

[0177] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS G12C comprising a nucleotidesequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in SEQ ID NO: 273. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0178] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cell-penetrating peptide), and b) a guide RNA targeting KRAS G12R comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 274-283. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 274, 277, or 279. In some embodiments, the guide RNA further comprising an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN- 106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0179] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS G12A comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 284-294. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 284, 285, or 290. In some embodiments, the guide RNA further comprising an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN- 106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA isbetween about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0180] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS G12S comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 295-304. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 295, 298, or 300. In some embodiments, the guide RNA further comprising an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN- 106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0181] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS G13D comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 305-309. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 305 or 308. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the firstand / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0182] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS G13C comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 310-315. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 310 and 313. In some embodiments, the guide RNA further comprising an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cellpenetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cellpenetrating peptide is an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN- 106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP)to the guide RNA is between about 1:1 and about 80: 1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0183] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS Q61H comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 316-322. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 316 or 321. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide(e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0184] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS Q61L comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 323-329. In some embodiments, the target sequence is set forth in any of SEQ ID NO: 323 or 328. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acidsequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0185] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS A18D comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 330-332. In some embodiments, the target sequence is set forth in SEQ ID NO: 332. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetratingpeptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0186] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS K117N comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 333-335. In some embodiments, the target sequence is set forth in SEQ ID NO: 333. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editingcomplex further comprises a DNA nuclease e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0187] In some embodiments, there is provided a genome-editing complex comprising a) a carrier (e.g., a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a first cellpenetrating peptide), and b) a guide RNA targeting KRAS A146T comprising a nucleotide sequence substantially complementary (such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence set forth in any of SEQ ID NOs: 336-341. In some embodiments, the target sequence is set forth in SEQ ID NO: 336 or 339. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx (e.g., Ava or a PEG moiety). In some embodiments, the first cell-penetrating peptide is an ADGN- 100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide). In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 89, 90, 270, 153-155, 434 and 435. In some embodiments, the guide RNA is complexed with the first cell-penetrating peptide. In some embodiments, the genome-editing complex further comprises a DNA nuclease (e.g., Cas9) or a polynucleotide encoding the DNA nuclease. In some embodiments, the complex comprises a second cell-penetrating peptide comprising an ADGN-100 peptide or ADGN-106 peptide (i.e., VEPEP-6 peptide) linked to a targeting moiety. In some embodiments, the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the CPP (the first and / or the second CPP) is complexed with a) the nucleotide sequence ending Cas polypeptide (e.g., Cas9) and / or b) the guide RNA. In some embodiments, the molar ratio of the cellpenetrating peptide (the first and / or the second CPP) to the guide RNA is between about 1 : 1 and about 80:1 (such as between about 5:1 and about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the cell-penetrating peptide (the first and / or the second CPP) to the polynucleotide encoding the Cas polypeptide is between about 1:1 and about 80:1 (such as between about 5:1 to about 20:1, such as between about 2:1 to about 50:1). In some embodiments, the molar ratio of the polynucleotide encoding the Cas polypeptide (e.g., a Cas mRNA) to the guide RNA is between about 1:10 and about 50:1 (such as between about 1:1 and about 10:1). In some embodiments, the molar ratio of the CPP:Cas mRNA:gRNA is about 20:1:1.

[0188] In some embodiments, there is provided a genome-editing complex comprising a) a first cell-penetrating peptide comprising an amino acid sequence set forth in SEQ ID NO: 434 or 435, b) a second CPP comprising an amino acid sequence set forth in SEQ ID NO: 272, 427, or 428 and b) a guide RNA targeting KRAS A146T comprising a nucleotide sequence 100% complementary to a target sequence set forth in any of SEQ ID NOs: 273-341. In some embodiments, the target sequence is set forth in SEQ ID NO: 274, 277, or 279. In some embodiments, the target sequence is set forth in SEQ ID NO: 284, 285, or 290. In some embodiments, the target sequence is set forth in SEQ ID NO: 295, 298, or 300. In some embodiments, the target sequence is set forth in SEQ ID NO: 305 or 308. In some embodiments, the target sequence is set forth in SEQ ID NO: 310 or 313. In some embodiments, the target sequence is set forth in SEQ ID NO: 316 or 321. In some embodiments, the target sequence is set forth in SEQ ID NO: 323 or 328. In some embodiments, the target sequence is set forth in SEQ ID NO: 332. In some embodiments, the target sequence is set forth in SEQ ID NO: 333. In some embodiments, the target sequence is set forth in SEQ ID NO: 336 or 339. In some embodiments, the complex further comprises apolynucleotide encoding a Cas nuclease (e.g., Cas9). In some embodiments, the ratio of CPP (both 1stCPP and 2ndCPP):polynucleotide encoding Cas nuclease:guide RNA is about 20:1:1.Carriers

[0189] A variety of delivery methods for cargo molecules, such as mRNA, have been developed, such as direct injection, lipid-based carriers, polymers and protein derivatives. The utility of lipid nanoparticles to deliver cargo molecules, such as mRNA, has been successfully demonstrated with COVID- 19 vaccines, such as mRNA- 1273 and BNT162b. It is expected that these vehicles such as lipid nanoparticles can successfully deliver the cargo molecules discussed here.

[0190] As provided herein, the compositions may include a transfer vehicle. As used herein, the terms “transfer vehicle,” “delivery vehicle,” “carrier” and the like refer to variant agents, pharmaceutical carriers, diluents, excipients and the like which are generally intended for use in connection with the administration of biologically active agents, including nucleic acids. The compositions and in particular the transfer vehicles described herein are capable of delivering mRNA to the target cell. In certain embodiments, the transfer vehicle is a lipid nanoparticle. In other embodiments, the transfer vehicle is a polymeric carrier, such as, e.g., polyethyleneimine. In some embodiments, the transfer vehicle comprises a cell-penetrating peptide.

[0191] In certain embodiments, the cargo molecules is an mRNA. In certain embodiments, the mRNA molecules of the application may be administered as naked or unpackaged mRNA. In some embodiments, the administration of the mRNA in the compositions of the application may be facilitated by inclusion of a suitable carrier. In certain embodiments, the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more mRNAs. As used herein, the terms “transfect” or “transfection” mean the intracellular introduction of an mRNA (e.g., a P53 mRNA) encoding a protein (e.g., a P53 protein) into a cell, and preferably into a target cell. The introduced mRNA may be stably or transiently maintained in the target cell. The term “transfection efficiency” refers to the relative amount of mRNA taken up by the target cell which is subject to transfection. In practice, transfection efficiency can be estimated by the amount of a reporter nucleic acid product expressed by the target cells following transfection. The mRNA in the compositions of the application may be introduced into target cells with or without a carrier or transfer vehicle.

[0192] In certain embodiments, the carriers employed in the compositions of the application may comprise a liposomal vesicle, or other means to facilitate the transfer of a cargo molecule to target cells and / or tissues. Preferred embodiments include compositions with high transfection efficacies and in particular those compositions that minimize adverse effects which are mediated by transfection of non-target cells. The compositions of the present application that demonstrate high transfection efficacies improve the likelihood that appropriate dosages of the cargo molecule will be delivered to the target cell, while minimizing potential systemic adverse effects.

[0193] The cargo molecule can be formulated with one or more acceptable reagents, which provide a vehicle for delivering such cargo molecule to target cells. Appropriate reagents are generally selected with regard to a number of factors, which include, among other things, the biological or chemical properties of the cargo molecule, the intended route of administration, the anticipated biological environment to which such cargo molecule will be exposed and the specific properties of the intended target cells. In some embodiments, transfer vehicles, such as liposomes, encapsulate the cargo molecule without compromising biological activity. In some embodiments, the transfer vehicle demonstrates preferential and / or substantial binding to a target cell relative to non-target cells. In a preferred embodiment, the transfer vehicle delivers its contents to the target cell such that the cargo molecule is delivered to the appropriate subcellular compartment, such as the cytoplasm.

[0194] In some embodiments, the compositions of the application employ a polymeric carrier alone or in combination with other carriers. Suitable polymers may include, for example, poly acrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, polyethylenimine (PEI), including, but not limited to branched PEI (25 kDa) and multi-domain-block polymers. Alternatively, suitable carriers include, but are not limited to, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, dry powders, nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, small-molecule targeted conjugates, andother vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable carrier. (Hum. Gene Ther. 2008 September; 19(9):887-95).Lipid Nanoparticles

[0195] In certain embodiments, the transfer vehicle in the compositions of the application is a liposomal transfer vehicle, e.g. a lipid nanoparticle or a lipidoid nanoparticle. In one embodiment, the transfer vehicle may be selected and / or prepared to optimize delivery of the cargo molecule to a target cell. For example, if the target cell is a hepatocyte the properties of the transfer vehicle (e.g., size, charge and / or pH) may be optimized to effectively deliver such transfer vehicle to the target cell, reduce immune clearance and / or promote retention in that target cell.

[0196] Liposomes (e.g., liposomal lipid nanoparticles) are known to be particularly for their use as transfer vehicles of diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16: 307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0197] In the context of the present application, a liposomal transfer vehicle typically serves to transport the cargo molecule to the target cell. For the purposes of the present application, the liposomal transfer vehicles are prepared to contain cargo molecule ( e.g., a cargo molecule) encoding a protein (e.g., a protein). The process of incorporation of the desired cargo molecule into a liposome is referred to as “loading” and is described in Lasic, et al., FEBS Lett., 312: 255-258, 1992. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome.

[0198] The purpose of incorporating a cargo molecule into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause therapid excretion of the nucleic acids. Accordingly, in a preferred embodiment of the present application, the selected transfer vehicle is capable of enhancing the stability of the cargo molecule contained therein. The liposome can allow the encapsulated cargo molecule to reach the target cell and / or may preferentially allow the encapsulated cargo molecule to reach the target cell, or alternatively limit the delivery of such cargo molecule to other sites or cells where the presence of the administered cargo molecule may be useless or undesirable. Furthermore, incorporating the cargo molecule into a transfer vehicle, such as for example, a cationic liposome, also facilitates the delivery of such cargo molecule into a target cell.

[0199] Ideally, liposomal transfer vehicles are prepared to encapsulate cargo molecule (e.g., a cargo molecule) encoding a protein (e.g., a protein) such that the compositions demonstrate high transfection efficiency and enhanced stability. While liposomes can facilitate introduction of nucleic acids into target cells, the addition of polycations e.g., poly L-lysine and protamine), as a copolymer can facilitate, and in some instances markedly enhance, the transfection efficiency of several types of cationic liposomes by 2-28 fold in a number of cell lines both in vitro and in vivo. (See N. J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.) Thus, in certain embodiments of the present application, the transfer vehicle is formulated as a lipid nanoparticle.

[0200] In certain embodiments, the cargo molecule (e.g., a cargo molecule) encoding a protein (e.g., a protein) is combined with a multi-component lipid mixture of varying ratios employing one or more cationic lipids, non-cationic lipids, helper lipids, and PEG-modified or PEGylated lipids designed to encapsulate various nucleic acid-based materials. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0201] Cationic lipids may include, but are not limited to ALNY-100 ((3aR,5s,6aS)-N,N- dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyetetrahydro-3aH-cyclopenta[d][l,3]dioxol-5- amine)), DODAP (l,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012 / 170889, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61 / 617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001(cis or trans) (Provisional Patent Application No. 61 / 617,468), aminoalcohol lipidoids such as those disclosed in W02010 / 053572, DOTAP (l,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O- octadecenyl- 3 -trimethylammonium propane), DLinDMA (l,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) (Heyes, et al., J. Contr. Rel. 107:276-287 (2005)), DLin-KC2-DMA (Semple, et al., Nature Biotech. 28:172-176 (2010)), C12-200 (Love, et al., Proc. Nat'l. Acad. Sci. 107:1864-1869 (2010)).

[0202] In some embodiments, DOTMA can be formulated alone or can be combined with the neutral lipid, DOPE (dioleoylphosphatidyl-ethanolamine), or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, DOGS (5-carboxyspermyl glycinedioctadecylamide), DOSPA (2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium) (Behr et al. Proc. Nat'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), DOTAP (1,2- Dioleoyl-3-Trimethylammonium- Propane). Contemplated cationic lipids also include DSDMA (l,2-distearyloxy-N,N-dimethyl-3-aminopropane, DODMA (l,2-dioleyloxy-N,N- dimethyl-3-aminopropane), DLenDMA ( 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane), DODAC (N-dioleyl-N,N-dimethylammonium chloride), DDAB (N,N-distearyl-N,N- dimethylammonium bromide), DMRIE (N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide), CLinDMA (3-dimethylamino-2-(cholest-5-en-3-beta- oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane), CpLinDMA (2-[5'-(cholest-5-en- 3-beta-oxy)-3'-oxapentoxy)-3-dimethy 1- l-(cis,cis-9', l-2'-octadecadienoxy)propane), DMOBA (N,N-dimethyl-3,4-dioleyloxybenzylamine), DOcarbDAP (1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane), DLinDAP (2,3-Dilinoleoyloxy-N,N- dimethylpropylamine), DLincarbDAP (l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane) , DLinCD AP ( 1 ,2-Dilinoleoylcarbamyl-3-dimethylaminopropane, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl-[l ,3]-dioxolane), DLin-K-XTC2- DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), or mixtures thereof.

[0203] Specific biodegradable lipids suitable for use in the compositions and methods of the application include:Compound 1and their salts.

[0204] Additional specific cationic lipids for use in the compositions and methods of the application are XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane) and MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate):MC3both of which are described in detail in US 20100267806.

[0205] Another cationic lipid that may be used in the compositions and methods of the application is NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-Nl,N16-diundecyl- 4,7, 10, 13-tetraazahexadecane- 1 , 16-diamide):which is described in WO06138380A2.

[0206] Suitable helper lipids include, but are not limited to DSPC (1,2-distearoyl-sn-glycero- 3 -phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2- dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine), DMPE (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol)), and cholesterol. Cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N,N- dimethyl-N-ethylcarboxamidocholesterol), 1 ,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6- methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate)(WO / 2011 / 068810).

[0207] Non-cationic lipids may also be used in the compositions of the application. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. “Anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, DSPC (distearoylphosphatidyl-choline), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidyl-choline), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidyl-glycerol), DOPE (dioleoylphosphatidylethanolamine), POPC (palmitoyloleoyl-phosphatidylcholine), POPE (palmitoyloleoyl-phosphatidylethanolamine), DOPE-mal (dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate), DDPE (dipalmitoyl phosphatidyl ethanolamine), DMPE (dimyristoylphosphoethanolamine), DSPE (distearoylphosphatidylethanolamine), SOPE (16-0- monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidy ethanolamine), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipid present in the transfer vehicle.

[0208] Polyethylene glycol (PEG)-modified phospholipids and derivatized lipids for use in nanoparticle formulations include, but are not limited to a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length, DMG- PEG2K, PEG-DSG, PEG-DMG, and PEG-derivatized ceramides (PEG-CER), including N- Octanoyl-Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000], (C8 PEG-2000 ceramide). The use of PEG-modified lipids is contemplated for use the compositions of the application, either alone or preferably in combination with other lipids which together comprise the transfer vehicle (e.g., a lipid nanoparticle). The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipids of the present application may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.

[0209] In addition, several reagents are commercially available to enhance transfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECT AMINE (DOSPA:DOPE) (Invitrogen), LIPGFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), and EFFECTENE.

[0210] Preferably, the transfer vehicle (e.g., a lipid nanoparticle) is prepared by combining multiple lipid and / or polymer components. For example, a transfer vehicle may comprise C12-200, DSPC, CHOL, and DMG-PEG or MC3, DSPC, chol, and DMG-PEG or C12-200, DOPE, chol, DMG-PEG2K. The selection of cationic lipids, non-cationic lipids and / or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the cargo molecule to be delivered. For example, a transfer vehicle may be prepared using C 12-200, DOPE, cholesterol, DMG- PEG2K at a molar ratio of 40:30:25:5; or DODAP, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18:56:20:6; or HGT5000, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 40:20:35:5; or HGT5001, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 40:20:35:5; or XTC, DSPC, cholesterol, PEG-DMG at a molar ratio of 57.5:7.5:31.5:3.5 or a molar ratio of 60:7.5:31:1.5; or MC3, DSPC, cholesterol, PEG-DMG in a molar ratio of 50:10:38.5:1.5 or a molar ratio of 40:15:40:5; or MC3, DSPC, cholesterol, PEG-DSG / GalNAc-PEGDSG in a molar ratio of 50:10:35:4.5:0.5; or ALNY-100, DSPC, cholesterol, PEG-DSG.

[0211] Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly. For example, in embodiments, the percentage of cationic lipid in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The percentage of non-cationic lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipid in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0212] In certain preferred embodiments, the lipid nanoparticles of the application comprise at least one of the following cationic lipids: XTC, MC3, NC98-5, ALNY-100, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In some embodiments, the transfer vehicle comprises cholesterol and / or a PEG-modified lipid. In some embodiments, the transfer vehicles comprise DMG-PEG2K.

[0213] The liposomal transfer vehicles for use in the compositions of the application can be prepared by various techniques which are presently known in the art. Multi-lamellar vesicles (MLV) may be prepared via conventional techniques, for example, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar vesicles (ULV) can then beformed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0214] In certain embodiments of this application, the compositions of the present application comprise a transfer vehicle wherein the cargo molecule is associated on both the surface of the transfer vehicle and encapsulated within the same transfer vehicle. For example, during preparation of the compositions of the present application, cationic liposomal transfer vehicles may associate with the cargo molecule through electrostatic interactions.

[0215] Selection of the appropriate size of a liposomal transfer vehicle must take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made. In some embodiments, it may be desirable to limit transfection of the cargo molecule to certain cells or tissues. For example, to target hepatocytes a liposomal transfer vehicle may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly, the liposomal transfer vehicle can readily penetrate such endothelial fenestrations to reach the target hepatocytes. Alternatively, a liposomal transfer vehicle may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposomal transfer vehicle may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomal transfer vehicle to hepatocytes. Generally, the size of the transfer vehicle is within the range of about 25 to 250 nm, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10 nm.

[0216] A variety of alternative methods known in the art are available for sizing of a population of liposomal transfer vehicles. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomal vesicles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes.Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.Cell-penetrating peptides

[0217] Cell Penetrating Peptides (CPP) are one of the promising non- viral strategies. Although definition of CPPs is constantly evolving, they are generally described as short peptides of less than 30 amino acids either derived from proteins or from chimeric sequences. They are usually amphipathic and possess a net positive charge (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206). CPPs are able to penetrate biological membranes, to trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, thereby facilitating interactions with the target. CPPs can be subdivided into two main classes, the first requiring chemical linkage with the cargo and the second involving the formation of stable, non-covalent complexes. CPPs from both strategies have been reported to favour the delivery of a large panel of cargos (plasmid DNA, oligonucleotide, siRNA, PNA, protein, peptide, liposome, nanoparticle...) into a wide variety of cell types and in vivo models (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206; Mickan et al. (2014) Curr Pharm Biotechnol 15, 200-209; Shukla et al. (2014) Mol Pharm 11, 3395-3408).

[0218] The concept of protein transduction domain (PTD) was initially proposed based on the observation that some proteins, mainly transcription factors, could shuttle within cells and from one cell to another (for review see Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195- 206). The first observation was made in 1988, by Frankel and Pabo. They showed that the transcription-transactivating (Tat) protein of HIV- 1 could enter cells and translocate into the nucleus. In 1991, the group of Prochiantz reached the same conclusions with the Drosophila Antennapedia homeodomain and demonstrated that this domain was internalized by neuronal cells. These works were at the origin of the discovery in 1994 of the first Protein Transduction Domain: a 16 mer-peptide derived from the third helix of the homeodomain of Antennapedia named Penetratin. In 1997, the group of Lebleu identified the minimal sequence of Tat required for cellular uptake, and the first proofs-of-concept of the application of PTD in vivo were reported by the group of Dowdy for the delivery of small peptides and large proteins (Gump JM, and Dowdy SF (2007) Trends Mol Med 13, 443-448.). Historically,the notion of Cell Penetrating Peptide (CPP) was introduced by the group of Langel, in 1998, with the design of the first chimeric peptide carrier, the Transportan, which derived from the N-terminal fragment of the neuropeptide galanin, linked to mastoparan, a wasp venom peptide. Transportan has been originally reported to improve the delivery of PNAs (peptide nucleic acids) both in cultured cells and in vivo (Langel U (2007) Handbook of Cell- Penetrating Peptides (CRC Taylor & Francis, Boca Raton)). In 1997, the group of Heitz and Divita proposed a new strategy involving CPP in the formation of stable but non-covalent complexes with their cargo (Morris et al. (1997) Nucleic Acids Res 25, 2730-2736). The strategy was first based on the short peptide carrier (MPG) consisting of two domains: a hydrophilic (polar) domain and a hydrophobic (apolar) domain. MPG was designed for the delivery of nucleic acids. The primary amphipathic peptide Pep-1 was then proposed for non- covalent delivery of proteins and peptides (Morris et al. (2001) Nat Biotechnol 19, 1173- 1176). Then the groups of Wender and of Futaki demonstrated that polyarginine sequences (Arg8) are sufficient to drive small and large molecules into cells and in vivo (Nakase et al. (2004) Mol Ther 10, 1011-1022; Rothbard et al. (2004) J Am Chem Soc 126, 9506-9507). Ever since, many CPPs derived from natural or unnatural sequences have been identified and the list is constantly increasing. Peptides have been derived from VP22 protein of Herpes Simplex Virus, from calcitonin, from antimicrobial or toxin peptides, from proteins involved in cell cycle regulation, as well as from polyproline-rich peptides (Heitz et al. (2009) Br J Pharmacol 157, 195-206). More recently, a new non-covalent strategy based on secondary amphipathic CPPs has been described. These peptides such as CADY and VEPEP-families are able to self-assemble in a helical shape with hydrophilic and hydrophobic residues on different side of the molecule. WO2014 / 053879 discloses VEPEP-3 peptides;WO2014 / 053881 discloses VEPEP-4 peptides; WO2014 / 053882 discloses VEPEP-5 peptides; W02012 / 137150 discloses VEPEP-6 peptides; W02014 / 053880 discloses VEPEP- 9 peptides; WO 2016 / 102687 discloses ADGN-100 peptides; US2010 / 0099626 discloses CADY peptides; and. U.S. Pat. No. 7,514,530 discloses MPG peptides; the disclosures of which are hereby incorporated herein by reference in their entirety.

[0219] The cell-penetrating peptides in the genome-editing complexes or nanoparticles of the present application are capable of forming stable complexes and nanoparticles with various molecules of a genome-editing system, such as nucleases (e.g., ZFNs, TALENs, and CRISPR-associated nucleases (such as Cas9 and Cpfl)), integrases (such as bacteriophage integrases, e.g., C31 ), and nucleic acids (e.g., guide RNAs, guide DNAs, and donor nucleicacids). Any of the cell-penetrating peptides in any of the genome-editing complexes or nanoparticles described herein may comprise or consist of any of the cell-penetrating peptide sequences described in this section.

[0220] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a cell-penetrating peptide selected from the group consisting of CADY, PEP-1, MPG, VEPEP-3 peptides, VEPEP-4 peptides, VEPEP-5 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the cell-penetrating peptide is present in a genome-editing complex. In some embodiments, the cell-penetrating peptide is present in a genome-editing complex present in the core of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRIS PR-associated endonuclease, such as Cas9). In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the cell-penetrating peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the surface layer of a nanoparticle. In some embodiments, the cell-penetrating peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent. WO2014 / 053879 discloses VEPEP-3 peptides; WO2014 / 053881 discloses VEPEP-4 peptides; WO2014 / 053882 discloses VEPEP-5 peptides; W02012 / 137150 discloses VEPEP-6 peptides;W02014 / 053880 discloses VEPEP-9 peptides; WO 2016 / 102687 discloses ADGN-100 peptides; US2010 / 0099626 discloses CADY peptides; and. U.S. Pat. No. 7,514,530 discloses MPG peptides; the disclosures of which are hereby incorporated herein by reference in their entirety.VEPEP-3 peptides

[0221] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a VEPEP-3 cell-penetrating peptide comprising the amino acid sequence X1X2X3X4X5X2X3X4X6X7X3X8X9X10X11X12X13 (SEQ ID NO: 44), wherein Xi is beta-A (“beta-alanine) or S, X2 is K, R or L (independently from each other), X3 is F or W (independently from each other), X4 is F, W or Y (independently from each other), X5 is E, R or S, Xe is R, T or S, X7 is E, R, or S, Xs is none, F or W, X9 is P or R, X10 is R or L, Xu isK, W or R, X12 is R or F, and X13 is R or K. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1X2WX4EX2WX4X6X7X3PRX11RX13 (SEQ ID NO: 45), wherein Xi is beta-A or S, X2 is K, R or L, X3 is F or W, X4 is F, W or Y, X5 is E, R or S, Xf> is R, T or S, X7 is E, R, or S, Xs is none, F or W, X9 is P or R, X10 is R or L, Xu is K, W or R, X12 is R or F, and X13 is R or K. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence XiKWFERWFREWPRKRR (SEQ ID NO: 46), XiKWWERWWREWPRKRR (SEQ ID NO: 47), XiKWWERWWREWPRKRK (SEQ ID NO: 48), XiRWWEKWWTRWPRKRK (SEQ ID NO: 49), or XiRWYEKWYTEFPRRRR (SEQ ID NO: 50), wherein Xi is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, wherein the cellpenetrating peptide is modified by replacement of the amino acid in position 10 by a nonnatural amino acid, addition of a non-natural amino acid between the amino acids in positions 2 and 3, and addition of a hydrocarbon linkage between the two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KX14WWERWWRX14WPRKRK (SEQ ID NO: 51), wherein Xi is beta-A or S and Xi4is a non-natural amino acid, and wherein there is a hydrocarbon linkage between the two non- natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1X2X3WX5X10X3WX6X7WX8X9X10WX12R (SEQ ID NO: 52), wherein Xi is beta- A or S, X2 is K, R or L, X3 is F or W, X5 is R or S, Xf> is R or S, X7 is R or S, Xs is F or W, X9 is R or P, X10 is L or R, and X12 is R or F. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence XiRWWRLWWRSWFRLWRR (SEQ ID NO: 53), XiLWWRRWWSRWWPRWRR (SEQ ID NO: 54), XiLWWSRWWRSWFRLWFR (SEQ ID NO: 55), or XiKFWSRFWRSWFRLWRR (SEQ ID NO: 56), wherein Xi is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 44 and 52-56, wherein the cell-penetrating peptide is modified by replacement of the amino acids in position 5 and 12 by non-natural amino acids, and addition of a hydrocarbon linkage between the two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWX14LWWRSWX14RLWRR (SEQ ID NO: 57), wherein Xi is a beta-alanine or a serine and X14 is a non-natural amino acid, and wherein there is a hydrocarbon linkage between the two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence beta-AKWFERWFREWPRKRR (SEQ ID NO: 58). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence beta- AKWWERWWREWPRKRR (SEQ ID NO: 59). In some embodiments, the VEPEP-3peptide comprises the amino acid sequence ASSLNIA-Ava-KWWERWWREWPRKRR (SEQ ID NO: 60). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence LSSRLDA-Ava-KWWERWWREWPRKRR (SEQ ID NO: 61). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence Ac-SYTSSTM-ava- KWWERWWREWPRKRR (SEQ ID NO: 62). In some embodiments, the VEPEP-3 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-3 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP- 3 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with a guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-3 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.VEPEP-6 peptides

[0222] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a VEPEP-6 cell-penetrating peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LX2RALWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 63), X1LX2LARWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 64) and X1LX2ARLWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 65), wherein Xi is beta-A or S, X2is F or W, X3is L, W, C or I, X4is S, A, N or T, X5is L or W, X6is W or R, X7is K or R, Xs is A or none, and X9 is R or S. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6X7X8 (SEQ ID NO: 66), wherein Xi is beta-A or S, X2is F or W, X3is L, W, C or I, X4is S, A, N or T, X5is L or W, X6is W or R, X7 is K or R, and Xs is A or none. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6KX7 (SEQ ID NO: 67), wherein Xi is beta-A or S, X2 is F or W, X3 is L or W, X4 is S, A or N, X5 is L or W, Xf> is W or R, X7 is A or none. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LFRALWRLLRX2LWRLLWX3(SEQ ID NO: 68), X1LWRALWRLWRX2LWRLLWX3A (SEQ ID NO: 69),X1LWRALWRLX4RX2LWRLWRX3A (SEQ ID NO: 70),X1LWRALWRLWRX2LWRLWRX3A (SEQ ID NO: 71),X1LWRALWRLX5RALWRLLWX3A (SEQ ID NO: 72), andX1LWRALWRLX4RNLWRLLWX3A (SEQ ID NO: 73), wherein Xi is beta-A or S, X2 is S or T, X3 is K or R, X4 is L, C or I and X5 ; L or I. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac- XiLFRALWRLLRSLWRLLWK-cysteamide (SEQ ID NO: 74), Ac- XiLWRALWRLWRSLWRLLWKA-cysteamide (SEQ ID NO: 75), Ac- XiLWRALWRLLRSLWRLWRKA-cysteamide (SEQ ID NO: 76), Ac- XiLWRALWRLWRSLWRLWRKA-cysteamide (SEQ ID NO: 77), Ac- XiLWRALWRLLRALWRLLWKA-cysteamide (SEQ ID NO: 78), and Ac- XiLWRALWRLLRNLWRLLWKA-cysteamide (SEQ ID NO: 79), wherein Xi is beta-A or S. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 63-79, further comprising a hydrocarbon linkage between two residues at positions 8 and 12. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac-XiLFRALWRsLLRSsLWRLLWK- cysteamide (SEQ ID NO: 80), Ac-XiLFLARWRsLLRSsLWRLLWK-cysteamide (SEQ ID NO: 81), Ac-XiLFRALWSsLLRSsLWRLLWK-cysteamide (SEQ ID NO: 82), Ac- XiLFLARWSsLLRSsLWRLLWK-cysteamide (SEQ ID NO: 83), Ac- XiLFRALWRLLRsSLWSsLLWK-cysteamide (SEQ ID NO: 84), Ac- XiLFLARWRLLRsSLWSsLLWK-cysteamide (SEQ ID NO: 85), Ac- XiLFRALWRLLSsSLWSsLLWK-cysteamide (SEQ ID NO: 86), Ac- XiLFLARWRLLSsSLWSsLLWK-cysteamide (SEQ ID NO: 87), and Ac- XiLFARsLWRLLRSsLWRLLWK-cysteamide (SEQ ID NO: 88), wherein Xi is beta-A or S and wherein the residues followed by an inferior "S" are those which are linked by said hydrocarbon linkage. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta-ALWRALWRLWRSLWRLLWKA (SEQ ID NO: 89). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs 90-117. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 90). In some embodiments, the VEPEP-6 peptide comprises a retro-inverso amino acid sequenceAKWLLRWLSRWLRWLARWLR (SEQ ID NO: 91). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-(PEG)7-PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 92) or Ac-(PEG)2-PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 93). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 94-103. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta- A- Ac-YIGSR-Ava- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 96). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta-A-Ac-YIGSR-Aun- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 98). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-YIGSR-Ahx- ALWRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 100) or Ac-YIGSR-Ahx- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 101). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence beta- Ac-GYVS-Ahx- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 102) or Ac-YIGSR- PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 103). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Stearyl-PA- ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 104). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 105-107. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence ALWRA(GalNac)LWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 111). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-SYTSSTM-ava- PALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 112). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac- THRPPNWSPVWPRALWRLWRSLWRLRWKA-NH2 (SEQ ID NO: 113). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac- CKTRRVPWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 114). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-CKTRRVP-ava- WRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 115). In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence Ac-CARPAR-ava- WRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 116). In some embodiments, the VEPEP- 6 peptide comprises an amino acid sequence Ac-THRPPNWSPV- ava- WRALWRLWRSLWRLRWK-NH2 (SEQ ID NO: 117). In some embodiments, the VEPEP- 6 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-6 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associatedwith a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-6 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-6 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.VEPEP-9 peptides

[0223] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a VEPEP-9 cell-penetrating peptide comprising the amino acid sequence X1X2X3WWX4X5WAX6X3X7X8X9X10X11X12WX13R (SEQ ID NO: 118), wherein Xi is beta- A or S, X2 is L or none, X3 is R or none, X4 is L, R or G, X5 is R, W or S, Xf> is S, P or T, X7 is W or P, Xs is F, A or R, X9 is S, L, P or R, X10 is R or S, Xu is W or none, X12 is A, R or none and X13 is W or F, and wherein if X3 is none, then X2, Xu and X12 are none as well. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence X1X2RWWLRWAX6RWX8X9X10WX12WX13R (SEQ ID NO: 119), wherein Xi is beta-A or S, X2 is L or none, Xf> is S or P, Xs is F or A, X9 is S, L or P, X10 is R or S, X12 is A or R, and X13 is W or F. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of XiLRWWLRWASRWFSRWAWWR (SEQ ID NO: 120), XiLRWWLRWASRWASRWAWFR (SEQ ID NO: 121), XiRWWLRWASRWALSWRWWR (SEQ ID NO: 122), XiRWWLRWASRWFLSWRWWR (SEQ ID NO: 123), XiRWWLRWAPRWFPSWRWWR (SEQ ID NO: 124), and XiRWWLRWASRWAPSWRWWR (SEQ ID NO: 125), wherein Xi is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of X1WWX4X5WAX6X7X8RX10WWR (SEQ ID NO: 126), wherein Xi is beta-A or S, X4 is R or G, X5 is W or S, X<> is S, T or P, X7 is W or P, Xs is A or R, and X10 is S or R. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of XiWWRWWASWARSWWR (SEQ ID NO: 127), XiWWGSWATPRRRWWR (SEQ ID NO: 128), and XiWWRWWAPWARSWWR (SEQ ID NO: 129), wherein Xi is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence beta-ALRWWLRWASRWFSRWAWWR (SEQ ID NO:130). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence KSYDTY-ava-ALRWLRWASRWFSRWAWR (SEQ ID NO: 131). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence ac- CKRAVRWWLRWASRWFSRWAWWR (SEQ ID NO: 132). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence beta-A-RWWLRWASRWFSRWAWR (SEQ ID NO: 133). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence KSYDTYAAETRRWASRWFSRWAWWR (SEQ ID NO: 134). In some embodiments, the VEPEP-9 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-9 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-9 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.ADGN-100 peptides

[0224] In some embodiments, a genome-editing complex or nanoparticle described herein comprises an ADGN-100 cell-penetrating peptide comprising the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 135), wherein Xi is any amino acid or none, and X2-X8 are any amino acid. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 136), wherein Xi is PA, S, or none, X2 is A or V, X3 is or L, X4 is W or Y, X5 is V or S, Xf> is R, V, or A, X7 is S or L, and Xs is W or Y. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence KWRSAGWRWRLWRVRSWSR (SEQ ID NO: 137), KWRSALYRWRLWRVRSWSR (SEQ ID NO: 138), KWRSALYRWRLWRSRSWSR (SEQ ID NO: 139), or KWRSALYRWRLWRSALYSR (SEQ ID NO: 140). In some embodiments, the ADGN-100 peptide comprises two residues separated by three or six residues that are linked by a hydrocarbon linkage. In some embodiments, the ADGN-100peptide comprises the amino acid sequence KWRSsAGWRsWRLWRVRSWSR (SEQ ID NO: 141), KWRsSAGWRWRsLWRVRSWSR (SEQ ID NO: 142), KWRSAGWRsWRLWRVRsSWSR (SEQ ID NO: 143), KWRSsALYRsWRLWRSRSWSR (SEQ ID NO: 144), KWRsSALYRWRsLWRSRSWSR (SEQ ID NO: 145), KWRSALYRsWRLWRSRsSWSR (SEQ ID NO: 146), KWRSALYRWRsLWRSsRSWSR (SEQ ID NO: 147), KWRSALYRWRLWRSsRSWSsR (SEQ ID NO: 148), KWRsSALYRWRsLWRSALYSR (SEQ ID NO: 149), KWRSsALYRsWRLWRSALYSR (SEQ ID NO: 150), KWRSALYRWRsLWRSsALYSR (SEQ ID NO: 151), or KWRSALYRWRLWRSsALYSsR (SEQ ID NO: 152), wherein the residues marked with a subscript “S” are linked by a hydrocarbon linkage. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 153-171. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of beta- AKWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 153). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of beta- AKWRSAGWRWRLWRVRSWSR (SEQ ID NO: 154) or beta- AKWRSALYRWRLWRVRSWSR (SEQ ID NO: 155). In some embodiments, the ADGN- 100 peptide comprises a retro-inverso amino acid sequence of RSWSRVRWLRWRWGASRWK (SEQ ID NO: 156). In some embodiments, the ADGN- 100 peptide comprises an amino acid sequence of Ac-(PEG)7-bA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 157) or beta- Ac-(PEG)2-pA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 158). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of Stearyl-PA- KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 159). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence of any one of SEQ ID NOS: 160-169. In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac- YIGSR-Ava-KWRSALWRWRLWRVRSWSR-NH2 (ava is a 5-amino pentanoic acid) (SEQ ID NO: 162). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac-YIGSR-Ahx-KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 167). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac-YIGSR- (PEG)n-pA-KWRSALWRWRLWRVRSWSR-NH2 (n = 2, 4, or 7) (SEQ ID NO: 170). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac- KWRSA(GALNAC)LWRWRLWRVRSWSR-NH2 (SEQ ID NO: 172). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence Ac- CARPARWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 173). In some embodiments,the ADGN-100 peptide comprises a core motif comprising an amino acid sequence of RWRLWRWSR (SEQ ID NO: 168). In some embodiments, the ADGN-100 peptide comprises an amino acid sequence TGNYKALHPDHNGWRSALRWRLWRWSR-NH2 (SEQ ID NO: 174) or Ac-TGNYKALHPDHNG-ava-WRSALRWRLWRWSR-NH2 (SEQ ID NO: 175). In some embodiments, the ADGN-100 peptide is present in a genome-editing complex. In some embodiments, the ADGN-100 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR- associated endonuclease, such as Cas9). In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the ADGN- 100 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the surface layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.VEPEP-4 peptides

[0225] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a VEPEP-4 cell-penetrating peptide comprising the amino acid sequence XWXRLXXXXXX (SEQ ID NO: 176), wherein X in position 1 is beta-A or S; X in positions 3, 9 and 10 are, independently from each other, W or F; X in position 6 is R if X in position 8 is S, and X in position 6 is S if X in position 8 is R; X in position 7 is L or none; X in position 11 is R or none, and X in position 7 is L if X in position 11 is none. In some embodiments, the VEPEP-4 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 177-180. In some embodiments, the VEPEP-4 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-4 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-4peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-4 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-4 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.VEPEP-5 peptides

[0226] In some embodiments, a genome-editing complex or nanoparticle described herein comprises a VEPEP-5 cell-penetrating peptide comprising the amino acid sequence RXWXRLWXRLR (SEQ ID NO: 181), wherein X in position 2 is R or S; and X in positions 4 and 8 are, independently from each other, W or F. In some embodiments, the VEPEP-5 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 182-187. In some embodiments, the VEPEP-5 peptide is present in a genome-editing complex. In some embodiments, the VEPEP-5 peptide is present in a genome-editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a DNA nuclease (such as a CRISPR-associated endonuclease, such as Cas9). In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with the guide RNA. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-5 peptide is present in an intermediate layer of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-5 peptide is linked to a targeting moiety. In some embodiments, the linkage is covalent.Cell-penetrating peptide modification

[0227] In some embodiments, the CPP described herein (e.g., VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) further comprises one or more moieties linked to (e.g., covalently linked to) the N-terminus of the CPP. In some embodiments, the one or more moieties is covalently linked to the N-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclearlocalization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, a linker moiety, and a targeting moiety. In some embodiments, the one or more moieties comprise an acetyl group covalently linked to the N-terminus of the CPP.

[0228] In some embodiments, the CPP described herein (e.g., VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) further comprises one or more moieties linked to (e.g., covalently linked to) the C-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of a cysteamide group, a cysteine, a thiol, an amide, a nitrilotriacetic acid, a carboxyl group, a linear or ramified Ci-Ce alkyl group, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, a linker moiety, and a targeting moiety. In some embodiments, the one or more moieties comprises a cysteamide group.

[0229] In some embodiments, the CPP described herein (e.g., PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) is stapled. “Stapled” as used herein refers to a chemical linkage between two residues in a peptide. In some embodiments, the CPP is stapled, comprising a chemical linkage between two amino acids of the peptide. In some embodiments, the two amino acids linked by the chemical linkage are separated by 3 or 6 amino acids. In some embodiments, two amino acids linked by the chemical linkage are separated by 3 amino acids. In some embodiments, the two amino acids linked by the chemical linkage are separated by 6 amino acids. In some embodiments, each of the two amino acids linked by the chemical linkage is R or S. In some embodiments, each of the two amino acids linked by the chemical linkage is R. In some embodiments, each of the two amino acids linked by the chemical linkage is S. In some embodiments, one of the two amino acids linked by the chemical linkage is R and the other is S. In some embodiments, the chemical linkage is a hydrocarbon linkage.

[0230] In some embodiments, the CPP is an L-peptide comprising L- amino acids. In some embodiments, the CPP is a retro-inverso peptide (e.g., a peptide made up of D-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds). In some embodiments, the retro- inverso peptide comprises a sequence of SEQ ID NO: 91 or 156.

[0231] In some embodiments, the CPP comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the cellpenetrating peptide.Targeting moiety

[0232] In some embodiments, the one or more moieties comprise a targeting moiety. In some embodiments, the targeting moiety is conjugated to the N-terminus of the CPP (e.g. an ADGN-100 peptide, e.g., a VEPEP-6 peptide). In some embodiments, the targeting moiety is conjugated to beta-alanine of an VEPEP-6 peptide (e.g., SEQ ID NO: 89 or 90). In some embodiments, the targeting moiety is conjugated to the C-terminus the CPP. In some embodiments, a first targeting moiety is conjugated to the N-terminus of the CPP and a second targeting moiety is conjugated to the C-terminus of the CPP.

[0233] In some embodiments, the targeting moiety comprises a targeting peptide that targets one or more organs. In some embodiments, the one or more organs are selected from the group consisting of muscle, heart, brain, spleen, lymph node, liver, lung, and kidney. In some embodiments, the targeting peptide targets brain. In some embodiments, the targeting peptide targets muscle. In some embodiments, the targeting peptide targets heart.

[0234] In some embodiments, the targeting moiety comprises at least about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises no more than about 8, 7, 6, 5, or 4 amino acids. In some embodiments, the targeting moiety comprises about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises a sequence selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, IGSR. In some embodiments, the sequence e.g., a targeting sequence) is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0235] In some embodiments, the targeting moiety comprises a targeting sequence selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the targeting moiety comprises a targeting sequence SYTSSTM (SEQ ID NO: 196). In some embodiments, the targeting moiety comprises a targeting sequence CKTRRVP (SEQ ID NO: 197). In some embodiments, the targeting moiety comprises a targeting sequence THRPPNWSPV (SEQ ID NO: 198). In some embodiments, the targeting moiety comprises a targeting sequence TGNYKALHPDHNG (SEQ ID NO: 199). In some embodiments, the targeting moiety comprises a targeting sequence CARPAR (SEQ ID NO: 200). In some embodiments, the targeting moiety comprises a targeting sequence ASSLNIA (SEQ ID NO:203). In some embodiments, the targeting moiety comprises a targeting sequence LSSRLDA (SEQ ID NO: 204). In some embodiments, the targeting moiety comprises a targeting sequence KSYDTY (SEQ ID NO: 205).

[0236] In some embodiments, the targeting moiety is conjugated to the CPP via a linker moiety such as any one of the linker moieties described herein.Linker moiety

[0237] In some embodiments, the one or more moieties comprise a linker moiety.

[0238] In some embodiments, the linker moiety comprises a polyglycine linker. In some embodiments, the linker comprises a P-Alanine. In some embodiments, the linker comprises at least about two, three, or four glycines, optionally continuous glycines. In some embodiments, the linker further comprises a serine. In some embodiments, the linker comprises a GGGGS or SGGGG sequence. In some embodiments, the linker comprises a Glycine-P-Alanine motif.

[0239] In some embodiments, the one or more moieties comprise a polymer (e.g., PEG, poly lysine, PET). In some embodiments, the polymer is conjugated to the N-terminus of the CPP. In some embodiments, the polymer is conjugated to the C-terminus of the CPP. In some embodiments, a first polymer is conjugated to the N-terminus of the CPP and a second polymer is conjugated to the C-terminus of the CPP. In some embodiments, the polymer is a PEG. In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG is a branched PEG. In some embodiments, the molecular weight of the PEG is no more than about 5 kDa, 10 kDa, 15kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is at least about 5 kDa, 10 kDa, 15kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa to about 10 kDa, about 10 kDa to about 15kDa, about 15 kDa to about 20 kDa, about 20kDa to about 30 kDa, or about 30 kDa to about 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of the PEG is selected from the group consisting of 5 kDa, 10 kDa, 20 kDa or 40 kDa. In some embodiments, the molecular weight of the PEG is about 5 kDa. In some embodiments, the molecular weight of the PEG is about 10 kDa. In some embodiments, the PEG comprises at least about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG consists of no more than about 10, 9, 8 or 7 ethylene glycol units. In some embodiments, the PEG consistsof about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG moiety consists of about one to eight, or about two to seven ethylene glycol units.

[0240] In some embodiments, the linker moiety is selected from the group consisting of beta alanine, cysteine, cysteamide bridge, poly glycine (such as G2 or G4), Aun (11-amino- undecanoic acid), Ava (5-amino pentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker moiety comprises Aun (11-amino-undecanoic acid). In some embodiments, the linker moiety comprises Ava (5-amino pentanoic acid). In some embodiments, the linker moiety comprises Ahx (aminocaproic acid).Carbohydrate moiety

[0241] In some embodiments, the cell-penetrating peptide further comprises a carbohydrate moiety. In some embodiments, the carbohydrate moiety is GalNAc. In some embodiments, the cell-penetrating peptide is an ADGN-106 peptide. In some embodiments, the cellpenetrating peptide is an ADGN-100 peptide. In some embodiments, the carbohydrate moiety modifies an alanine within the cell-penetrating peptide. In some embodiments, the cellpenetrating peptide is set forth in SEQ ID NO: 111 or 172.Cell-penetrating peptide mixture

[0242] In some embodiments, the cell-penetrating peptide in the genome-editing complexes is a mixture of a) a first peptide comprising a first cell-penetrating peptide (such as any of the cell-penetrating peptide described herein); b) a second peptide comprising a second cellpenetrating peptide (such as any of the cell-penetrating peptide described herein), wherein the second peptide comprises a polyethylene glycol (PEG) moiety that is covalently linked to the second cell-penetrating peptide, and wherein the first peptide does not have a PEG moiety. In some embodiments, the first and / or the second cell-penetrating peptide is a PTD-based peptide, an amphipathic peptide, a poly-arginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first and the second cell-penetrating peptides are selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-4 peptides, VEPEP-5 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the molar ratio of the cell-penetrating peptide to the cargo (such as the guide RNA optionally with a DNA nuclease or a nucleotide encoding the DNA nuclease) is between about 1:1 and about 100:1 (such as about between about 1:1 and about 50:1, or about 2:1 to about 50:1). In some embodiments, the average diameter of the genome-editing complex is between about 20 nm and about 1000 nm (such as about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm). In some embodiments, the PEG moiety consists of about one to ten (such as about 1-8, 2-7, 1-5, or 6-10) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 0.5 kDa (such as about 0.05-0.1, 0.05-0.4, 0.1-0.3, 0.05-0.25, 0.25-0.5 kDa). In some embodiments, the PEG moiety is conjugated to the N- or C-terminus of the second cell-penetrating peptide. In some embodiments, the PEG moiety is conjugated to a site within the second cellpenetrating peptide.

[0243] In some embodiments, the ratio of the first cell-penetrating peptide to the second cellpenetrating peptide is about 20:1 to about 1:1 (such as about 15:1 to about 2:1, about 10:1 to about 4:1).

[0244] In some embodiments, the first and / or the second cell-penetrating peptides are selected from VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, the first and / or the second cell-penetrating peptide are selected from VEPEP-6 peptides, and ADGN-100 peptides.

[0245] In some embodiments, the PEG moiety is a linear PEG. In some embodiments, the PEG moiety is a branched PEG.Cargo molecules

[0246] In some embodiments, cell-penetrating peptides described herein are complexed with the one or more cargo molecules. In some embodiments, the cell-penetrating peptides are non-covalently complexed with at least one of the one or more cargo molecules. In some embodiments, the cell-penetrating peptides are non-covalently complexed with each of the one or more cargo molecules. In some embodiments, the cell-penetrating peptides are covalently complexed with at least one of the one or more cargo molecule. In some embodiments, the cell-penetrating peptides are covalently complexed with each of the one or more cargo molecules.

[0247] As described above, the genome-editing complex or nanoparticles described herein comprise a guide RNA as described above. In some embodiments, the genome-editing complex or nanoparticle comprises one or more genome-editing molecules (such as a DNA nuclease or a polynucleotide encoding the DNA nuclease).

[0248] The genome-editing complex or nanoparticle described herein comprises a guide RNA that targets a mutated KRAS, such as any of the guide RNA described in the “Guide RNAs” section above.DNA nuclease

[0249] In some embodiments, the complex or nanoparticle described herein further comprises a DNA nuclease or a nucleotide encoding the DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR- associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activatorlike effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.

[0250] For example, in some embodiments, a complex or nanoparticle described herein comprises an RGEN (e.g., Cas9). In some embodiments, the protein or polypeptide is between about 10 kDa and about 200 kDa (such as about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa, including any ranges between these values). In some embodiments, the complex or nanoparticle comprises a plurality of proteins or polypeptides, wherein each of the plurality of protein or polypeptides is between about 10 kDa and about 200 kDa (such as about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa, including any ranges between these values).

[0251] In some embodiments, a complex or nanoparticle described herein further comprises a nucleic acid encoding a DNA nuclease. In some embodiments, the nucleic acid is between about 20 nt and about 20 kb (such as about any of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any ranges between these values). In some embodiments, the nucleic acid is DNA, such as a DNA plasmid encoding a genome-editing system molecule. In some embodiments, the DNA plasmid comprises an expression cassette for expressing the genome-editing system molecule. In some embodiments, the DNA plasmid is between about 1 kb and about 20 kb (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any ranges between these values). In some embodiments, the nucleic acid is RNA, such as mRNA encoding a genome-editing system molecule. In some embodiments, the mRNA is between about 100 nt and about 10 kb (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 kb, including any ranges between these values).

[0252] In some embodiments, the complex or nanoparticle comprises a plurality of nucleic acids, such as any of the nucleic acids described herein. For example, in some embodiments, the complex or nanoparticle comprises a gRNA and a nucleic acid encoding a genomeediting system molecule (e.g., a DNA plasmid or mRNA encoding the DNA nuclease). In some embodiments, the complex or nanoparticle comprises nucleic acid encoding a plurality of genome-editing system molecules (e.g., one or more DNA plasmid encoding the plurality of genome-editing system molecules, or a plurality of mRNAs encoding the plurality of genome-editing system molecules).

[0253] In some embodiments, the nucleic acids are single stranded oligonucleotides. In some embodiments, the nucleic acids are double stranded oligonucleotides. The nucleic acids described herein may be any of a range of length of up to, but not necessarily 200 nucleotides in the case of antisense oligonucleotides, RNAi, siRNA, shRNA, iRNA, antagomirs or up to 1000 kilo bases in the case of plasmid DNA.

[0254] In some embodiments, the nucleic acids are plasmid DNA or DNA fragments (for example DNA fragments of lengths of up to about 1000 bp). In addition, the plasmid DNA or DNA fragments may be hypermethylated or hypomethylated. In some embodiments, the plasmid DNA or DNA fragments encode one or more genes, and may contain regulatory elements necessary for the expression of said one or more genes. In some embodiments, the plasmid DNA or DNA fragments may comprise one or more genes that encode a selectable marker, allowing for maintenance of the plasmid DNA or DNA fragment in an appropriate host cell.CRISPR-associated nuclease

[0255] In some embodiments, the DNA nuclease is a CRISPR-associated nuclease. In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171:3553-3556

[1989] ), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See,Groenen et al., Mol. Microbiol., 10:1057-1065

[1993] ; Hoe et al., Emerg. Infect. Dis., 5:254- 263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] ; and Mojica et al., Mol. Microbiol., 36:244-246

[2000] ). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al.,

[2000] , supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella. Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0256] In some embodiments, the DNA nuclease comprises a CRIS PR-associated nuclease, e.g., a Cas protein that is an RNA guided nuclease. The Cas protein is a nuclease that binds to a guide RNA which recognizes a target sequence. The Cas protein may a single or double strand into the target site. CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; and class 2 is divided into types II, V, and VI. Different Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Casio, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7. See, e.g., Jinek et al., Science (2012) 337 (6096):816- 821; Dang et al., Genome Biology (2015) 16:280; Ran et al., Nature (2015) 520:186-191;Zetsche et al., Cell (2015) 163:759-771; Strecker et al., Nature Comm. (2019) 10:212; and Yan et al., Science (2019) 363:88-91. The most widely used Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from S. pyogenes or S. aureus.

[0257] The type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs), each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as “protospacer adjacent motifs” (PAMs).

[0258] While the foregoing description has focused on Cas9 nuclease, it should be appreciated that other RNA-guided nucleases exist which utilize gRNAs that differ in some ways from those described to this point. For instance, Cpfl (CRISPR from Prevotella and Franciscella 1; also known as Cas 12a) is an RNA-guided nuclease that only requires a crRNA and does not need a tracrRNA to function.

[0259] Alternatively, synthetic gRNAs gRNAs can be single guide RNAs (sgRNAs such as any of those described above) composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest. The tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA. One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.

[0260] In some embodiments, Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and / or other characteristics. For example, the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects(e.g., eSpCas9, SpCas9-HFl, FlypaSpCas9, FleFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9). In some embodiments, the Cas nuclease has one or more inactive nuclease domains.

[0261] In some embodiments, the genome editing enzyme is an RNA guided nuclease. In some embodiments, the RNA guided nuclease is selected from the group consisting of Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas 10, Cas 12, Cas 12a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxll, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof. In some embodiments, an RNA guided nuclease is fused to a cytosine deaminase, an adenosine deaminase, or a reverse transcriptase.

[0262] In some embodiments, the DNA nuclease is a Cas protein. Non-limiting examples of Cas proteins include Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cpfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, such as inducible, inactivated, or split Cas proteins (see for example Dominguez et al. (2015). Nature Reviews Molecular Cell Biology; Polstein, L. R., & Gersbach, C. A. (2015). Nature chemical biology, 11(3): 198-200; Dow et al. (2015). Nature biotechnology, 33(4):390-394; Zetsche et al. (2015). Nature biotechnology, 33(2): 139-142; Kleinstiver et al. (2015). Nature. 523:481-485; Bikard et al. (2013). Nucleic acids research, 41(15):7429-7437; Qi et al. (2013). Cell, 152(5): 1173-1183). These enzymes are known to those of skill in the art; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2, and the amino acid sequence of Acidaminococcus sp. Cpfl protein may be found in the SwissProt database under accession number U2UMQ6.

[0263] In some embodiments, the DNA nuclease comprises an unmodified or modified CRISPR enzyme that has DNA cleavage activity, such as Cas9. In some embodiments, theCRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme is Cpfl, and may be Cpfl from Acidaminococcus or Lachnospiraceae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, the CRISPR enzyme is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickase may be used in combination with guide sequences, e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.

[0264] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form. Other mutations may be useful; where the Cas9 or other CRISPR enzyme is from a species other than S. pyogenes, mutations in corresponding amino acids may be made to achieve similar effects.

[0265] In some embodiments, the Cas protein (such as Cas9) is a split Cas protein comprising an N-terminal Cas protein fragment, Cas(N), and a C-terminal Cas protein fragment, Cas(C), wherein Cas(N) is fused to a first dimerization domain and Cas(C) is fused to a second dimerization domain, and wherein the first and second dimerization domains facilitate dimerization of Cas(N) and Cas(C) to form a complex with a functional Cas nuclease activity. In some embodiments, dimerization of the first and second dimerizationdomains is sensitive to a dimerization agent. For example, in some embodiments, the first and second dimerization domains comprise the FK506 binding protein 12 (FKBP) and FKBP rapamycin binding (FRB) domains of the mammalian target of rapamycin (mTOR), and the dimerization agent is rapamycin.

[0266] In some embodiments, the complex or nanoparticle described herein comprises a polynucleotide encoding a CRISPR enzyme (such as Cas9 endonuclease) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0267] In some embodiments, the CRISPR enzyme described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 212); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 213)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 214) or RQRRNELKRSP (SEQ ID NO: 215); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 216); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 217) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 218) and PPKKARED (SEQ ID NO: 219) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 220) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 221) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 222) and PKQKKRK (SEQ ID NO: 223) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 224) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 225) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 226) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 227) of the steroid hormone receptors (human) glucocorticoid.

[0268] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Nonlimiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, betaglucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0269] In some embodiments, the cargo comprises a base editor. Base editors have been developed that convert Cas endonucleases into programmable nucleotide deaminases, thus facilitating the introduction of C-to-T mutations (by C-to-U deamination) or A-to-G mutations (by A-to-I deamination) without induction of a double-strand break. Base editors comprise a nickase form of SpCas9 (nSpCas9, to stimulate cellular DNA mismatch repair) fused to a nucleobase deaminase enzyme as well as an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI). See Rees et al., Nature Communications Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery volume 8, Article number: 15790 (2017); Komor et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016).

[0270] In some embodiments, the cargo comprises a prime editor. Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and provide the new geneticinformation to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. See Anzalone et al. Search-and-replace genome editing without doublestrand breaks or donor DNA. Nature volume 576, pagesl49-157(2019).

[0271] In some embodiments, the cargo comprises a fusion protein comprising a catalytically disabled nuclease (such as a catalytically disabled Cas9 endonuclease) and a reversed transcriptase (such as a pentamutant of M-MLV reverse transcriptase). See for example, Anzalone & Liu et al., Nature. 2019 Dec; 576 (7785):149-157. In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein.

[0272] In some embodiments, the cargo comprises a fusion protein comprising a catalytically disabled nuclease (such as a catalytically disabled Cas9 endonuclease) and a nucleobase deaminase enzyme. In some embodiments, the nucleobase deaminase enzyme is APOBEC1 cytidine deaminase. In some embodiments, the nucleobase deaminase enzyme is cytidine deaminase CDA1. In some embodiments, the fusion protein further comprises a DNA glycosylase inhibitor. In some embodiments, the DNA glycosylase inhibitor is uracil DNA glycosylase inhibitor (UGI). In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein.ZFPs and ZFNs; TALs, TALEs, and TALENs

[0273] In some embodiments, the cargo molecule includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease (or nucleic acid encoding the DNA-binding protein / effector protein fusion). Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Fronteirs in Immunology, 4(221), 1-7 (2013). In some embodiments, the guide RNA described herein can be in the form of a DNA (i.e., guide DNA, gDNA) encoding the RNA that guides ZFP or TAL to the target set.ZFPs and ZFNs

[0274] In some embodiments, the cargo molecule comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds DNA in a sequencespecific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

[0275] Among the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers.

[0276] ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632- 637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054;7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties.

[0277] In some embodiments, the cargo molecule includes a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN). In some embodiments, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from the Type IIS restriction endonuclease Fok I. Fok I generally catalyzes double- stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982.]

[0278] In some embodiments, ZFNs target a gene present in a target cell. In some aspects, the ZFNs efficiently generate a double strand break (DSB), for example at a predetermined site in the coding region of the gene. Typical regions targeted include exons, regions encoding N- terminal regions, first exon, second exon, and promoter or enhancer regions. In some embodiments, transient expression of the ZFNs promotes highly efficient and permanent disruption of the target gene in target cells. In particular, in some embodiments, delivery of the ZFNs results in the permanent disruption of the gene with efficiencies surpassing 50%.

[0279] Many gene-specific engineered zinc fingers are available commercially. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a platform (CompoZr) for zinc- finger construction in partnership with Sigma-Aldrich (St. Louis, MO, USA), allowing investigators to bypass zinc-finger construction and validation altogether, and provides specifically targeted zinc fingers for thousands of proteins. Gaj et al., Trends in Biotechnology , 2013, 31(7), 397-405. In some embodiments, commercially available zinc fingers are used or are custom designed. (See, for example, Sigma- Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).TALEs and TALENs

[0280] In some embodiments, the cargo molecule includes a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein.

[0281] A TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Each TALE repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Diresidue (RVD), typically at positions 12 and / or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T and non-canonical (atypical) RVDs are also known. See, U.S. Patent Publication No. 20110301073. In some embodiments, TALEs may be targeted to any gene by design of TAL arrays with specificity to the target DNA sequence. The target sequence generally begins with a thymidine.

[0282] In some embodiments, the cargo molecule includes a DNA binding endonuclease, such as a TALE-nuclease (TALEN). In some aspects the TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain to cleave a nucleic acid target sequence. In some embodiments, the TALE DNA-binding domain has been engineered to bind a target sequence within a target gene.

[0283] In some embodiments, the TALEN recognizes and cleaves the target sequence in the gene. In some aspects, cleavage of the DNA results in double- stranded breaks. In some aspects the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. In some aspects, repair mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct;23(10):394-8) or via the so-called microhomology- mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene. In some embodiments, the modification may be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and / or selected by well-known methods in the art.

[0284] In some embodiments, TALE repeats are assembled to specifically target a gene. (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology . 31, 251-258 (2013)). Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0285] In some embodiments the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker which provides for identification and / or selection of cells which received said vector.Meganucleases

[0286] In some embodiments, the DNA nuclease comprises a meganuclease. Meganucleases are enzymes in the endonuclease family that are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). Meganucleases are grouped into families based on their structural motifs, which affect nuclease activity and / or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLID ADG family, which owe their name to a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774. On the other hand, the GIY- YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which arerequired for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811. The His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774. Members of the NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.

[0287] Because the chance of identifying a natural meganuclease for a particular target DNA sequence is low due to the high specificity requirement, various methods including mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Chevalier et al., Mol. Cell. (2002) 10:895-905; Epinat et al., Nucleic Acids Res (2003) 31 :2952-2962; Silva et al., J Mol. Biol. (2006) 361 :744-754; Seligman et al., Nucleic Acids Res (2002) 30:3870-3879; Sussman et al., J Mol Biol (2004) 342:31-41 ; Doyon et al., J Am Chem Soc (2006) 128:2477-2484; Chen et al., Protein Eng Des Sei (2009) 22:249-256; Arnould et al., J Mol Biol. (2006) 355:443-458; Smith et al., Nucleic Acids Res. (2006) 363(2) :283-294.

[0288] Like ZFNs and TALENs, Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11 :11 -27.Transposases

[0289] In some embodiments, the DNA nuclease comprises a transposase. Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. By linking transposases to other systems such as the CRISPR / Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA. There are two known DNA integration methods using transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons. The transposase-dependent DNA integrationdoes not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.Donor nucleic acid

[0290] In some embodiments, the complex or nanoparticles described herein further comprises a donor nucleic acid. In some embodiments, the donor nucleic acid is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex. A donor nucleic acid may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the donor nucleic acid comprises a sequence that is complementary to a portion of a polynucleotide comprising the target sequence. In some embodiments, when a donor nucleic acid and a polynucleotide comprising a target sequence are optimally aligned, the donor nucleic acid overlaps with one or more nucleotides of the target sequence (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a donor nucleic acid and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the donor nucleic acid in the region of complementarity is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.RNAi targeting mutant form of KRAS

[0291] In some embodiments, the cargo further comprises one or more RNAi e.g., siRNA) that targets a mutant form of KRAS. In some embodiments, the mutant form of KRAS comprises an aberration of KRAS, wherein the aberration of KRAS comprises a mutation on codon 12, 13, 17, 18, 34, 59, 61, 68, 95, 96, 117, and / or 146 of KRAS. In some embodiments, the aberration of KRAS comprises a mutation on codon 12, or 61 of KRAS. In some embodiments, the aberration of KRAS is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, GBR, G13A, G13D, G13V, GBP, S17G, A18D, P34S, A59T, Q61H, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, R68M, R68S, H95D, H95Q, H95R, Y96C, Y96D, K117N, A146P, A146T and A146V. In some embodiments, the aberration of KRAS is selected from the group consisting of G12C, G12S, GBR, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, GBP, S17G, P34S, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the aberration of KRAS is selected from the group consisting of G12C, GBR, G12S, G12A, G12D, G12V, G13C,G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T and A146V. In some embodiments, the aberration of KRAS is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, GBR, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the aberration of KRAS is selected from the group consisting of KRAS G12C, G12D, GBR, G12S, G12V and G13D. In some embodiments, the aberration of KRAS is selected from G12C, G12D and Q61K.

[0292] In some embodiments, the one or more RNAi (e.g., siRNA) is selected from the group consisting of SEQ ID NOs: 228-234.Modifications

[0293] In some embodiments, a complex or nanoparticle as described herein comprises a targeting moiety, wherein the targeting moiety is a ligand capable of cell-specific and / or nuclear targeting. A cell membrane surface receptor and / or cell surface marker is a molecule or structure which can bind said ligand with high affinity and preferably with high specificity. Said cell membrane surface receptor and / or cell surface marker is preferably specific for a particular cell, i.e. it is found predominantly in one type of cell rather than in another type of cell (e.g. galactosyl residues to target the asialoglycoprotein receptor on the surface of hepatocytes). The cell membrane surface receptor facilitates cell targeting and internalization into the target cell of the ligand (e.g. the targeting moiety) and attached molecules (e.g. the complex or nanoparticle of the application). A large number of ligand moieties / ligand binding partners that may be used in the context of the present application are widely described in the literature. Such a ligand moiety is capable of conferring to the complex or nanoparticle of the application the ability to bind to a given binding-partner molecule or a class of binding-partner molecules localized at the surface of at least one target cell. Suitable binding-partner molecules include without limitation polypeptides selected from the group consisting of cell-specific markers, tissue-specific markers, cellular receptors, viral antigens, antigenic epitopes and tumor-associated markers. Binding-partner molecules may moreover consist of or comprise, for example, one or more sugar, lipid, glycolipid, antibody molecules or fragments thereof, or aptamer. According to the application, a ligand moiety may be for example a lipid, a glycolipid, a hormone, a sugar, a polymer (e.g. PEG, polylysine, PET), an oligonucleotide, a vitamin, an antigen, all or part of a lectin, all or part of a polypeptide, such as for example JTS1 (WO 94 / 40958), an antibody or a fragment thereof, or a combination thereof. In some embodiments, the ligand moiety used in the present application is a peptideor polypeptide having a minimal length of 7 amino acids. It is either a native polypeptide or a polypeptide derived from a native polypeptide. “Derived” means containing (a) one or more modifications with respect to the native sequence (e.g. addition, deletion and / or substitution of one or more residues), (b) amino acid analogs, including non-naturally occurring amino acids, (c) substituted linkages, or (d) other modifications known in the art. The polypeptides serving as ligand moiety encompass variant and chimeric polypeptides obtained by fusing sequences of various origins, such as for example a humanized antibody which combines the variable region of a mouse antibody and the constant region of a human immunoglobulin. In addition, such polypeptides may have a linear or cyclized structure (e.g. by flanking at both extremities a polypeptide ligand by cysteine residues). Additionally, the polypeptide in use as a ligand moiety may include modifications of its original structure by way of substitution or addition of chemical moieties (e.g. glycosylation, alkylation, acetylation, amidation, phosphorylation, addition of sulfhydryl groups and the like). The application further contemplates modifications that render the ligand moiety detectable. For this purpose, modifications with a detectable moiety can be envisaged (i.e. a scintigraphic, radioactive, or fluorescent moiety, or a dye label and the like). Such detectable labels may be attached to the ligand moiety by any conventional techniques and may be used for diagnostic purposes (e.g. imaging of tumoral cells). In some embodiments, the binding-partner molecule is an antigen (e.g. a target cell-specific antigen, a disease-specific antigen, an antigen specifically expressed on the surface of engineered target cells) and the ligand moiety is an antibody, a fragment or a minimal recognition unit thereof (e.g. a fragment still presenting an antigenic specificity) such as those described in detail in immunology manuals (see for example Immunology, third edition 1993, Roitt, Brostoff and Male, ed Gambli, Mosby). The ligand moiety may be a monoclonal antibody. Many monoclonal antibodies that bind many of these antigens are already known, and using techniques known in the art in relation to monoclonal antibody technology, antibodies to most antigens may be prepared. The ligand moiety may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example, ScFv). In some embodiments, the ligand moiety is selected among antibody fragments, rather than whole antibodies. Effective functions of whole antibodies, such as complement binding, are removed. ScFv and dAb antibody fragments may be expressed as a fusion with one or more other polypeptides. Minimal recognition units may be derived from the sequence of one or more of the complementary-determining regions (CDR) of the Fv fragment. Whole antibodies, and F(ab')2 fragments are “bivalent”. By “bivalent” it is meant that said antibodies and F(ab')2 fragments have two antigen binding sites. In contrast, Fab,Fv, ScFv, dAb fragments and minimal recognition units are monovalent, having only one antigen binding sites. In some embodiments, the ligand moiety allows targeting to a tumor cell and is capable of recognizing and binding to a molecule related to the tumor status, such as a tumor- specific antigen, a cellular protein differentially or over-expressed in tumor cells or a gene product of a cancer-associated vims. Examples of tumor- specific antigens include but are not limited to MUC-1 related to breast cancer (Hareuven i et al., 990, Eur. J. Biochem 189, 475-486), the products encoded by the mutated BRCA1 and BRCA2 genes related to breast and ovarian cancers (Miki et al, 1994, Science 226, 66-7 1; Fuireal et al, 1994, Science 226, 120- 122; Wooster et al., 1995, Nature 378, 789-792), APC related to colon cancer (Poiakis, 1995, Curr. Opin. Genet. Dev. 5, 66-71), prostate specific antigen (PSA) related to prostate cancer, (Stamey et al., 1987, New England J. Med. 317, 909), carcinoma embryonic antigen (CEA) related to colon cancers (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738- 2748), tyrosinase related to melanomas (Vile et al, 1993, Cancer Res. 53, 3860-3864), receptor for melanocyte- stimulating hormone (MSH) which is highly expressed in melanoma cells, ErbB-2 related to breast and pancreas cancers (Harris et al., 1994, Gene Therapy 1, 170-175), and alpha- foetoprotein related to liver cancers (Kanai et al., 1997, Cancer Res. 57, 46 1-465). In some embodiments, the ligand moiety is a fragment of an antibody capable of recognizing and binding to the MUC-1 antigen and thus targeting MUC-1 positive tumor cells. In some embodiments, the ligand moiety is the scFv fragment of the SM3 monoclonal antibody which recognizes the tandem repeat region of the MUC- 1 antigen (Burshell et al. , 1987, Cancer Res. 47, 5476-5482; Girling et al., 1989, Int. J. Cancer 43, 1072-1076; Dokumo et al., 1998, J. Mol. Biol. 284, 713-728). Examples of cellular proteins differentially or overexpressed in tumor cells include but are not limited to the receptor for interleukin 2 (IL- 2) overexpressed in some lymphoid tumors, GRP (Gastrin Release Peptide) overexpressed in lung carcinoma cells, pancreas, prostate and stomach tumors (Michael et al., 1995, Gene Therapy 2, 660-668), TNF (Tumor Necrosis Factor) receptor, epidermal growth factor receptors, Fas receptor, CD40 receptor, CD30 receptor, CD27 receptor, OX-40, a-v integrins (Brooks et al, 994, Science 264, 569) and receptors for certain angiogenic growth factors (Hanahan, 1997, Science 277, 48). Based on these indications, it is within the scope of those skilled in the art to define an appropriate ligand moiety capable of recognizing and binding to such proteins. To illustrate, IL-2 is a suitable ligand moiety to bind to TL-2 receptor. In the case of receptors that are specific to fibrosis and inflammation, these include the TGFbeta receptors or the Adenosine receptors that are identified above and are suitable targets for application compositions. Cell surface markers for multiple myeloma include, but are notlimited to, CD56, CD40, FGFR3, CS1, CD138, IGF1R, VEGFR, and CD38, and are suitable targets for application compositions. Suitable ligand moieties that bind to these cell surface markers include, but are not limited to, anti-CD56, anti-CD40, PRO-001, Chir-258, HuLuc63, anti-CD138-DMl, anti-IGFIR and bevacizumab.Nanoparticles

[0294] The present application in one aspect provides a nanoparticle comprising a core comprising any one or more of genome-editing complexes described above.

[0295] In some embodiments, there is provided a nanoparticle comprising a core comprising a genome-editing complex described herein, wherein the cell-penetrating peptide in the genome-editing delivery complex is associated with the cargo. In some embodiments, the association is non-covalent. In some embodiments, the association is covalent.

[0296] In some embodiments, the nanoparticle further comprises a surface layer (e.g., a shell) comprising a peripheral cell-penetrating peptide (z.e., CPP), wherein the core is coated by the shell. In some embodiments, the peripheral CPP is the same as a CPP in the core. In some embodiments, the peripheral CPP is different than any of the CPPs in the core. In some embodiments, the peripheral CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a poly-arginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-4, VEPEP-5, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the peripheral CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN- 100 peptide. IN some embodiments, the peripheral cell-penetrating peptide is selected from the group consisting of PEP- 1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides. In some embodiments, at least some of the peripheral cell-penetrating peptides in the surface layer are linked to a targeting moiety. In some embodiments, the linkage is covalent. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by genetic methods. In some embodiments, the nanoparticle further comprises an intermediate layer between the core of the nanoparticle and the surface layer. In some embodiments, the intermediate layer comprises an intermediate CPP. In some embodiments, the intermediate CPP is the same as a CPP in the core. In some embodiments, the intermediate CPP is different than any of the CPPs in the core. In some embodiments, the intermediate CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a poly-arginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the intermediate CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide.

[0297] In some embodiments, the nanoparticle comprises two or more guide RNAs such as any one of the guide RNAs described herein. In some embodiments, the two or more guide RNAs targets two or more different KRAS mutations. In some embodiments, the two or more different KRAS mutations are selected from the group consisting of G12D, G12V, and G12C. In some embodiments, the two or more guide RNAs are contained in the same genomeediting complex. In some embodiments, the two or more guide RNAs are contained in different genome-editing complex.

[0298] In some embodiments, the nanoparticle core comprises a plurality of genome-editing complexes. In some embodiments, the nanoparticle core comprises a plurality of genomeediting complexes present in a predetermined ratio. In some embodiments, the predetermined ratio is selected to allow the most effective use of the nanoparticle in any of the methods described below in more detail. In some embodiments, the nanoparticle core further comprises one or more additional guide RNAs, one or more additional cell-penetrating peptides, one or more additional genome-editing nucleases, and / or one or more additional donor nucleic acids.

[0299] In some embodiments, the one or more additional genome-editing complex comprises at least one or more (e.g., two, three, four, five, six, seven, or eight) of the guide RNAs that targets a different KRAS mutation. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12C (such as any one of the guide RNA targeting G12C described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12D, G12V, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T (such as any one of the guide RNA including those targeting G12V G12D described herein e.g., in sequence listing). In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12D (such as any one of the guide RNA targeting G12D described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, thenanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12V (such as any one of the guide RNA targeting G12V described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12D, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12R (such as any one of the guide RNA targeting G12R described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12D, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12A (such as any one of the guide RNA targeting G12A described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12D, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G12S (such as any one of the guide RNA targeting G12S described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12D, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G13D (such as any one of the guide RNA targeting G13D described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G12D, G13C, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting G13C (such as any one of the guide RNA targeting G13C described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G12D, Q61H, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting Q61H (such as any one of the guide RNA targetingQ61H described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A,G12S, G13D, G13C, G12D, Q61L, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting Q61L (such as any one of the guide RNA targeting Q61L described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G13C, G12D, Q61H, A18D, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting A18D (such as any one of the guide RNA targeting A18D described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G13C, G12D, Q61L, Q61H, K117N, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting KI 17N (such as any one of the guide RNA targeting KI 17N described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G13C, G12D, Q61L, A18D, Q61H, or A146T. In some embodiments, the nanoparticle described herein comprises a) a first genome-editing complex comprising a first guide RNA that specifically targeting A146T (such as any one of the guide RNA targeting A146T described herein), and b) a second genome-editing complex comprising a second guide RNA that specifically targeting another mutation selected from G12C, G12V, G12R, G12A, G12S, G13D, G13C, G12D, Q61L, A18D, K117N, or Q61H.

[0300] In some embodiments, the nanoparticle further comprises one or more additional cellpenetrating peptides. In some embodiments, the one or more additional cell-penetrating peptides include, but are not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (such as a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, at least some of the one or more additional cellpenetrating peptides are linked to a targeting moiety. In some embodiments, the linkage is covalent.

[0301] In some embodiments, according to any of the nanoparticles described herein, the mean size (diameter) of the nanoparticle is from about 20 nm to about 1000 nm, including for example from about 50 nm to about 800 nm, from about 75 nm to about 600 nm, from about 100 nm to about 600 nm, and from about 200 nm to about 400 nm. In some embodiments, themean size (diameter) of the nanoparticle is no greater than about 1000 nanometers (nm), such as no greater than about any of 900, 800, 700, 600, 500, 400, 300, 200, or 100 nm. In some embodiments, the average or mean diameter of the nanoparticle is no greater than about 200 nm. In some embodiments, the average or mean diameters of the nanoparticles is no greater than about 150 nm. In some embodiments, the average or mean diameter of the nanoparticle is no greater than about 100 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 20 nm to about 400 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 30 nm to about 400 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 40 nm to about 300 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 50 nm to about 200 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 60 nm to about 150 nm. In some embodiments, the average or mean diameter of the nanoparticle is about 70 nm to about 100 nm. In some embodiments, the nanoparticles are sterile-filterable.

[0302] In some embodiments, the zeta potential of the nanoparticle is from about -30 mV to about 60 mV (such as about any of -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mV, including any ranges between these values). In some embodiments, the zeta potential of the nanoparticle is from about -30 mV to about 30 mV, including for example from about -25 mV to about 25 mV, from about -20 mV to about 20 mV, from about -15 mV to about 15 mV, from about -10 mV to about 10 mV, and from about -5 mV to about 10 mV. In some embodiments, the polydispersity index (PI) of the nanoparticle is from about 0.05 to about 0.6 (such as about any of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6, including any ranges between these values). In some embodiments, the nanoparticle is substantially non-toxic.Compositions

[0303] In some embodiments, there is provided a composition (e.g., a pharmaceutical composition) comprising a genome-editing complex or nanoparticle as described herein. In some embodiments, the composition is a pharmaceutical composition comprising a genomeediting complex or nanoparticle as described herein and a pharmaceutically acceptable diluent, excipient, and / or carrier.

[0304] In some embodiments, the composition comprises a mixture of two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations. For example, in some embodiments, the compositioncomprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12V. In some embodiments, the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12C. In some embodiments, the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12C, and b) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12V. In some embodiments, the composition comprises a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D, b) a third nanoparticle comprising a second guide RNA that specifically targets KRAS G12V, c) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12C.

[0305] In some embodiments, the concentration of the complex or nanoparticle in the composition is from about 1 nM to about 100 mM, including for example from about 10 nM to about 50 mM, from about 25 nM to about 25 mM, from about 50 nM to about 10 mM, from about 100 nM to about 1 mM, from about 500 nM to about 750 pM, from about 750 nM to about 500 pM, from about 1 pM to about 250 pM, from about 10 pM to about 200 pM, and from about 50 pM to about 150 pM. In some embodiments, the pharmaceutical composition is lyophilized.

[0306] The term “pharmaceutically acceptable diluent, excipient, and / or carrier” as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals. The term diluent, excipient, and / or “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like, including lyophilization aids. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. Examples of suitable pharmaceutical diluent, excipient, and / or carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulation should suit the mode of administration. The appropriate diluent, excipient, and / or carrier will be evident to those skilled in the art and will depend in large part upon the route of administration.

[0307] In some embodiments, a composition comprising a genome-editing complex or nanoparticle as described herein further comprises a pharmaceutically acceptable diluent, excipient, and / or carrier. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier affects the level of aggregation of a genome-editing complex or nanoparticle in the composition and / or the efficiency of intracellular delivery mediated by a genome-editing complex or nanoparticle in the composition. In some embodiments, the extent and / or direction of the effect on aggregation and / or delivery efficiency mediated by the pharmaceutically acceptable diluent, excipient, and / or carrier is dependent on the relative amount of the pharmaceutically acceptable diluent, excipient, and / or carrier in the composition.

[0308] For example, in some embodiments, the presence of a pharmaceutically acceptable diluent, excipient, and / or carrier (such as a salt, sugar, chemical buffering agent, buffer solution, cell culture medium, or carrier protein) at one or more concentrations in the composition does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that does not promote and / or contribute to aggregation of the genomeediting complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 150% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 100% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 50% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 20% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 15% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises the pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 10% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a salt, including, without limitation, NaCl. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a sugar, including, without limitation, sucrose, glucose, and mannitol. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a chemical buffering agent, including, without limitation, HEPES. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a buffer solution, including, without limitation, PBS. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a cell culture medium, including, withoutlimitation, DMEM. Particle size can be determined using any means known in the art for measuring particle size, such as by dynamic light scattering (DLS). For example, in some embodiments, an aggregate having a Z-average as measured by DLS that is 10% greater than the Z-average as measured by DLS of a genome-editing complex or nanoparticle is 10% larger than the genome-editing complex or nanoparticle.

[0309] In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 100% (such as no more than about any of 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 75% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 50% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 20% larger than the size of the genomeediting complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 15% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 10% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the concentration of the salt in the composition is no more than about 100 mM (such as no more than about any of 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM, including any ranges between any of these values). In some embodiments, the salt is NaCl.

[0310] In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 25% (such as no more than about any of 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 75% larger than the size of the genomeediting complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 50% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 20% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 15% larger than the size of the genomeediting complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 10% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the concentration of the sugar in the composition is no more than about 20% (such as no more than about any of 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values). In some embodiments, the sugar is sucrose. In some embodiments, the sugar is glucose. In some embodiments, the sugar is mannitol.

[0311] In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes tothe formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 10% (such as no more than about any of 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 7.5% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 5% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 3% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genomeediting complex or nanoparticles having a size no more than about 1% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a chemical buffering agent (e.g., HEPES or phosphate) at a concentration that does not promote and / or contribute to the formation of aggregates of the genome-editing complex or nanoparticles. In some embodiments, the chemical buffering agent is HEPES. In some embodiments, the HEPES is added to the composition in the form of a buffer solution comprising HEPES. In some embodiments, the solution comprising HEPES has a pH between about 5 and about 9 (such as about any of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, including any ranges between these values). In some embodiments, the composition comprises HEPES at a concentration of no more than about 75 mM (such as no more than about any of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 mM or less, including any ranges between any of these values). In some embodiments, the chemical buffering agent is phosphate. In some embodiments, the phosphate is added to the composition in the form of a buffer solution comprising phosphate. In some embodiments, the composition does not comprise PBS.

[0312] In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 150% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 100% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g. , DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 50% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 25% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 10% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the cell culture medium is DMEM. In some embodiments, the composition comprises DMEM at a concentration of no more than about 70% (such as no more than about any of 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10%, or less, including any ranges between any of these values).

[0313] In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that does not promote and / or contribute to aggregation of the genome-editing complex or nanoparticle, or promotes and / or contributes to the formation of aggregates of theI llgenome-editing complex or nanoparticles having a size no more than about 200% (such as no more than about any of 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, including any ranges between any of these values) larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 150% larger than the size of the genomeediting complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 100% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 50% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genome-editing complex or nanoparticles having a size no more than about 25% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genomeediting complex or nanoparticles having a size no more than about 10% larger than the size of the genome-editing complex or nanoparticle. In some embodiments, the carrier protein is albumin. In some embodiments, the albumin is human serum albumin.

[0314] In some embodiments, a pharmaceutical composition as described herein is formulated for intravenous, intratumoral, intraarterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intracerebroventricular, intrathecal, intravesicular, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavity, or oral administration, or nebulization (NB) or intratracheal instillation.

[0315] Exemplary dosing frequencies include, but are not limited to, no more than once every three days.Methods of preparation

[0316] In some embodiments, there is provided a method of preparing a genome-editing complex or nanoparticle as described herein.

[0317] In some embodiments, there is provided a method of preparing the genome-editing complex comprising a peptide and a cargo molecule (e.g., a guide RNA) as described above, comprising combining the peptide with the cargo molecule, thereby forming the genomeediting complex.

[0318] In some embodiments, there is provided a method of preparing the genome-editing complex comprising a first cell-penetrating peptide and a second cell-penetrating peptide as described above, comprising a) combining the first cell-penetrating peptide and the second cell-penetrating peptide, thereby forming a peptide mixture; b) combining the peptide mixture with the cargo, thereby forming the genome-editing complex.

[0319] In some embodiments, the peptide or the peptide mixture and the cargo molecule are combined at a molar ratio from about 1:1 to about 100:1 (such as about between about 1:1 and about 50:1, such as about 2:1 to about 50:1), respectively.

[0320] In some embodiments, the method comprises mixing a first solution comprising the cargo molecule with a second solution comprising the peptide or peptide mixture to form a third solution, wherein the third solution comprises or is adjusted to comprise i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS, and wherein the third solution is incubated to allow formation of the genome-editing complex. In some embodiments, the first solution comprises the cargo in sterile water and / or wherein the second solution comprises the peptide or peptide mixture in sterile water. In some embodiments, the third solution is adjusted to comprise i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS after incubating to form the genome-editing complex.

[0321] In some embodiments, the method further comprises a filtration process, wherein the genome-editing complex is filtered through a pore-sized membrane. In some embodiments, the pore has a diameter of at least about 0.1 m (such as at least about 0.1 pm, 0.15 pm, 0.2 pm, 0.25 pm, 0.3 pm, 0.35 pm, 0.4 pm, 0.45 pm, 0.5pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1.0 pm, 1.1 pm or 1.2 pm). In some embodiments, the pore has a diameter of no more about 1.2 pm, 1.0 pm, 0.8 pm, 0.6 pm, 0.5 pm, 0.45 pm, 0.4 pm, 0.35 pm, 0.3 pm, or 0.25 pm. In some embodiments, the port has a diameter of about 0.1 pm to about 1.2 pm (such as about 0.1 to about 0.8 pm, about 0.2 to about 0.5 pm).

[0322] In some embodiments, for a stable composition comprising a cargo molecule delivery complex or nanoparticle of the application, the average diameter of the complex ornanoparticle does not change by more than about 10%, and the poly dispersity index does not change by more than about 10%.

[0323] Also provided are methods of preparing any of the peptides comprising cellpenetrating peptides described herein.Method of use (e.g., method of treatment)

[0324] The present application in one aspect provides a method of treating a disease (such as a cancer) in an individual comprising administering to the individual a genome-editing complex or nanoparticle comprising a guide RNA as described above. The present application in another aspect provides a method of modifying mutated KRAS in a cell comprising contacting the cell with the genome-editing complex or nanoparticle comprising a guide RNA as described above.

[0325] In some embodiments, there is provided a method of treating a cancer (such as a pancreatic cancer, colorectal cancer or a lung cancer) in an individual comprising administering to the individual a genome-editing complex or nanoparticle comprising a guide RNA described herein (e.g., a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341). In some embodiments, the genome-editing complex or nanoparticle is intravenously administered to the individual.

[0326] In some embodiments, there is provided a method of modifying mutated KRAS in a cell, comprising contacting the cell with a genome-editing complex or nanoparticle comprising a guide RNA targeting mutated KRAS comprising a guide RNA described herein (e.g., a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341). In some embodiments, the genome-editing complex or nanoparticle is intravenously administered to the individual.

[0327] In some embodiments, there is provided a method of treating a cancer that has a KRAS G12C mutation, wherein the method comprises administering a genome-editing complex comprising a) a guide RNA comprising a nucleotide sequence 100% complementary to a target sequence set forth in SEQ ID NOs: 273; b) a cell-penetrating peptide, wherein the cellpenetrating peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, 153-175, 259-270, 272, 353-355, 367-377, 382-383, 387- 396, 418-422, and 427-434 (e.g., SEQ ID Nos 89, 90, 162, 270, 355, 427-434), and c) a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the DNAnuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the complex comprises a) a first CPP comprising an amino acid sequence set forth in any of SEQ ID NO: 89, 90, 270, 153-155, 434 and 435, optionally wherein the first CPP comprises an amino acid sequence set forth in SEQ ID NO: 434 or 435, and b) a second CPP comprising an amino acid sequence set forth in any of 427-433, optionally wherein the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the DNA nuclease is a fusion protein, wherein the fusion protein further comprises a second enzyme that will allow base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the genome-editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA further comprising an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified (e.g., 5-Methoxyuridine). In some embodiments, the DNA nuclease is a Cas9 or Casl2a polynucleotide.

[0328] In some embodiments, there is provided a method of treating a cancer that has a KRAS G12R mutation, wherein the method comprises administering a genome-editing complex comprising a) a guide RNA comprising a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 274-283 (e.g., SEQ ID NO: 274, 277, or 279); b) a cell-penetrating peptide, wherein the cell-penetrating peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 89- 107, 111-117, 153-175, 259-270, 272, 353-355, 367-377, 382-383, 387-396, 418-422, and 427-434 (e.g., SEQ ID Nos 89, 90, 162, 270, 355, 427-434), and c) a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the complex comprises a) a first CPP comprising an amino acid sequence set forth in any of SEQ ID NO: 89, 90, 270, 153-155, 434 and 435, optionally wherein the first CPP comprises an amino acid sequence set forth in SEQ ID NO: 434 or 435, and b) a second CPP comprising an amino acid sequence set forth in any of 427-433, optionally wherein the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429. In some embodiments, the DNA nuclease is a fusion protein, wherein the fusion protein further comprises a second enzyme that will allow baseediting or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the genome-editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, t...

Claims

CLAIMS1. A non-naturally occurring polynucleotide comprising a guide RNA for targeting mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341.

2. The non-naturally occurring polynucleotide of claim 1, wherein the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 273-341.

3. The non-naturally occurring polynucleotide of claim 1 or claim 2, wherein: a) the guide RNA specifically targets G12C, wherein the target sequence is set forth in SEQ ID NO: 273; b) the guide RNA specifically targets G12R, wherein the target sequence is set forth in any one of SEQ ID NOs: 274-283, optionally wherein the target sequence is set forth in SEQ ID NO: 274, 277, or 279; c) the guide RNA specifically targets G12A, wherein the target sequence is set forth in any one of SEQ ID NOs: 284-294, optionally wherein the target sequence is set forth in SEQ ID NO: 284, 285, or 290; d) the guide RNA specifically targets G12S, wherein the target sequence is set forth in any one of SEQ ID NOs: 295-304, optionally wherein the target sequence is set forth in SEQ ID NO: 295, 298, or 300; e) the guide RNA specifically targets G13D, wherein the target sequence is set forth in any one of SEQ ID NOs: 305-309, optionally wherein the target sequence is set forth in SEQ ID NO: 305 or 308; f) the guide RNA specifically targets G13C, wherein the target sequence is set forth in any one of SEQ ID NOs: 310-315, optionally wherein the target sequence is set forth in SEQ ID NO: 310 or 313; g) the guide RNA specifically targets Q61H, wherein the target sequence is set forth in any one of SEQ ID NOs: 316-322, optionally wherein the target sequence is set forth in SEQ ID NO: 316 or 321; h) the guide RNA specifically targets Q61L, wherein the target sequence is set forth in any one of SEQ ID NOs: 323-329, optionally wherein the target sequence is set forth in SEQ ID NO: 323 or 328;i) the guide RNA specifically targets A18D, wherein the target sequence is set forth in any one of SEQ ID NOs: 330-332, optionally wherein the target sequence is set forth in SEQ ID NO: 332; j) the guide RNA specifically targets KI 17N, wherein the target sequence is set forth in any one of SEQ ID NOs: 333-335, optionally wherein the target sequence is set forth in SEQ ID NO: 333; or k) the guide RNA specifically targets A146T, wherein the target sequence is set forth in any one of SEQ ID NOs: 336-341, optionally wherein the target sequence is set forth in SEQ ID NO: 336 or 339.

4. The non-naturally occurring polynucleotide of any one of claims 1-3, wherein the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).

5. The non-naturally occurring polynucleotide of any one of claims 1-4, wherein the polynucleotide a) is chemically modified (e.g., 5-Methoxyuridine), and / or b) comprises prime editing guide RNA (pegRNA), optionally wherein the pegRNA comprises a prime editing site and a reverse transcriptase template.

6. A genome-editing complex comprising a) a polynucleotide of any one of claims 1-5, and b) a carrier, wherein the carrier promotes the delivery of the polynucleotide to a cell.

7. The genome-editing complex of claim 6, further comprising a DNA nuclease or a polynucleotide encoding the DNA nuclease.

8. The genome-editing complex of claim 7, wherein the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.

9. The genome-editing complex of claim 8, wherein the DNA nuclease comprises a Cas polypeptide.

10. The genome-editing complex of claim 8 or claim 9, wherein the Cas polypeptide a) is or comprises a Cas9 or a modified Cas9, and / or b) is fused with a deaminase.

11. The genome-editing complex of claim 10, optionally wherein the deaminase is a cytosine base editor or an adenine base editor, further optionally wherein the cytosine base editor is BE3, BE4, or A3A-PBE, and the adenine base editor is ABE7.10 or ABE8e.

12. The genome-editing complex of any one of claims 7-11, wherein the carrier comprises a lipid, a polymer, a viral vector, a extracellular vesicle, an exosome, a cellpenetrating peptide (CPP).

13. The genome-editing complex of claim 12, wherein the carrier comprises a CPP.

14. The genome-editing complex of claim 13, wherein the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptides, PEP-2 peptides, PEP-3 peptides, VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, and ADGN-100 peptides.

15. The genome-editing complex of claim 13 or claim 14, wherein the cell-penetrating peptide further comprises one or more moieties covalently linked to N-terminus of the first cell-penetrating peptide, and wherein the one or more moieties are selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety, optionally wherein: a) the cell-penetrating peptide comprises an acetyl group covalently linked to the N- terminus of the first cell-penetrating peptide; b) the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide, further optionally wherein the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205, 235-240, 351-352, 356-366, 378-386, 397-407, and 423-426.

16. The genome-editing complex of any one of claims 13-15, wherein the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a poly glycine linker moiety, a PEG moiety, Aun, Ava, and Ahx, optionally wherein the linker moiety is a PEG moiety or Ava, further optionally wherein the PEG moiety comprises any of two to twelve, two to ten, two to seven and two to three ethylene glycol units.

17. The genome-editing complex of any one of claims 13-16, wherein the cell-penetrating peptide comprises, from N-terminus, an acetyl group, a targeting moiety and a linker moiety covalently linked to the N-terminus of the cell-penetrating peptide.

18. The genome-editing complex of any one of claims 13-17, wherein the cell-penetrating peptide a) further comprises a carbohydrate moiety, optionally wherein the carbohydrate moiety is GalNAc, b) is a retro-inverso peptide, and / or c) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, 153-175, 259-270, 272, 353-355, 367-377, 382-383, 387-396, 418-422, and 427-434, optionally wherein the CPP comprises an amino acid selected from the group consisting of 89, 90, 162, 270, 355, 427- 434.

19. The genome-editing complex of any one of claims 13-18, wherein the molar ratio of the cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1, optionally wherein the molar ratio of the cell-penetrating peptide to the guide RNA is about 20:1.

20. The genome-editing complex of any one of claims 13-19, wherein the complex comprises a polynucleotide encoding a Cas nuclease, and wherein the molar ratio of the cellpenetrating peptide to the polynucleotide encoding the DNA nuclease is between about 1 : 1 and about 80:1, further wherein the Cas nuclease is a Cas9 or a modified Cas9, further wherein the molar ratio of the cell-penetrating peptide to the polynucleotide encoding the DNA nuclease is 20:1.

21. The genome-editing complex of any one of claims 7-20, further comprising one or more additional guide RNAs comprising different guide sequences, optionally wherein at least two of the two or more guide RNAs target one single KRAS mutation, further optionally wherein at least two of the two or more guide RNAs target two or more different KRAS mutations, further optionally wherein at least two of the two or more guide RNAs target G12D, G12V, G12C, G12R, G12A, G12S, G13D, G13C, Q61H, Q61L, A18D, K117N, or A146T.

22. The genome-editing complex of any one of claims 7-21, wherein the average diameter of the genome-editing complex is between about 10 nm and about 300 nm.

23. The genome-editing complex of any one of claims 7-22, wherein the complex comprises a) a first CPP comprising an amino acid sequence set forth in any of SEQ ID NO:89, 90, 270, 153-155, 434 and 435, optionally wherein the first CPP comprises an amino acid sequence set forth in SEQ ID NO: 434 or 435, and b) a second CPP comprising an amino acid sequence set forth in any of 427-433, optionally wherein the second CPP comprises an amino acid sequence set forth in SEQ ID NO: 427, 428 or 429.

24. A nanoparticle comprising a core comprising the genome-editing complex of any one of claims 1-23.

25. A pharmaceutical composition comprising the guide RNA of any one of claims 1-6, the genome-editing complex of any one of claims 7-23, or the nanoparticle of claim 24, and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein the composition comprises two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.

27. A method of preparing the genome-editing complex of any one of claims 7-23, comprising combining the first cell-penetrating peptide with the guide RNA, thereby forming the genome-editing complex.

28. A method of modifying mutated KRAS in a cell, comprising contacting the cell with guide RNA of any one of claims 1-6, the genome-editing complex of any one of claims 7-23, or the nanoparticle of claim 24, optionally wherein the method results in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 20%-about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in the cell.

29. A method of delivering a guide RNA to a cell, comprising contacting the cell with guide RNA of any one of claims 1-6, the genome-editing complex of any one of claims 7-23, or the nanoparticle of claim 24, optionally wherein the method results in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 10% to about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in the cell.

30. A method of treating a cancer in an individual comprising administering the individual an effective amount of the pharmaceutical composition of claim 25 or 26.

31. The method of claim 30, wherein:1) the individual comprises a secondary mutation in KRAS, optionally wherein the secondary mutation comprises a R68, Y96, or A59 mutation in KRAS, optionally the individual comprises a R68M, Y96D, or A59T mutation;2) the cancer comprises a copy number variation in KRAS;3) the cancer comprises an upregulated KRAS mRNA level and / or KRAS protein relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy;4) the cancer comprises a mutation in KRAS promoter that increases the strength of the promoter; and / or5) the cancer comprises an increased wildtype RAS signaling relative to a corresponding tissue or organ in a reference individual, a non-cancer tissue or organ in the same individual, or the same cancer prior to a prior therapy, optionally wherein the cancer has an increased level of active GTP-bound wildtype RAS, optionally wherein the wildtype RAS comprises H-RAS and / or N-RAS.

32. The method of claim 30 or claim 31, wherein the individual has been subjected to a KRAS inhibitor treatment.

33. The method of claim 32, wherein the cancer is resistant, refractory or recurrent to the KRAS inhibitor, further optionally the individual developed a secondary mutation after the KRAS inhibitor treatment.

34. The method of claim 32 or claim 33, wherein: a) the KRAS inhibitor specifically binds to the mutant KRAS protein, and / or b) the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC- 9805, sotorasib, adagrasib, ganetespib, RMC-6236, YL- 17231, BDTX-4933, QTX3034, ABT-200, ADT-1004, AN9025, OC211, JAB-23425, BI-2865, BI-2493, ABREV01, A2A- 03, LY3537982, and LY-4066434, optionally wherein the KRAS inhibitor is selected from the group consisting of MRTX1133, RMC-9805, sotorasib, adagrasib, and ganetespib.

35. The method of any one of claims 30-34, wherein the individual does not develop a secondary KRAS mutation in any of the exons after the KRAS treatment.

36. The method of any one of claims 30-35, further comprising administering a second agent.

37. The method of any one of claims 30-36, , wherein the method results in indel frequences of at least 10%, 20%, 30%, 40%, 50%, or 60% (e.g., about 20%-about 70%, about 50% to about 80 %, about 50% to about 70%, or about 60% to about 70%) in cancer cells harboring a KRAS mutation (i.e., the KRAS mutation the guide RNA targets).

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